CN109988234A

CN109988234A - 1 catenin of yeast recombination human source type i collagen α, synthetic method and its application

Info

Publication number: CN109988234A
Application number: CN201910126233.9A
Authority: CN
Inventors: 梁亮; 张永正
Original assignee: Jiangsu Yuezhi Biology Medicine Co ltd
Current assignee: Jiangsu Yuezhi Biology Medicine Co ltd
Priority date: 2019-02-20
Filing date: 2019-02-20
Publication date: 2019-07-09
Also published as: CN110964099A

Abstract

The invention discloses 1 catenin of a primary yeast recombination human source type i collagen α, synthetic method and its applications, 1 catenin of recombination human source type i collagen α of the invention, it is made of N-terminal affinity purification label, 1 chain maturation peptide sequence of source of people I-type collagen α and C-terminal affinity purification label, the bispecific affinity purification label of design at its both ends, the purifying for being conducive to the recombinant protein is also beneficial to its detection and overall length identification.The present invention is synthesized using the usual codon optimization glue protogene sequence of Pichia pastoris and artificial full genome at the genetic level first, then recombinant vector is built by PCR, digestion, connection etc., and be integrated into yeast chromosomal, it is built into the recombinant yeast pichia pastoris engineering bacteria of 1 catenin of excreting and expressing recombinant human source type i collagen α.The bispecific affinity purification label that 1 catenin of recombination human source type i collagen α of the invention carries, can carry out the purifying of two step specificity, the product of readily available high-purity.

Description

1 catenin of yeast recombination human source type i collagen α, synthetic method and its application

Technical field

The present invention relates to technical field of bioengineering, specifically a kind of 1 catenin of yeast recombination human source I Collagen Type VI α is closed At method and its application.

Background technique

Collagen is the most abundant class protein family of animal in-vivo content, accounts for about the 30% of animal body total protein Left and right.Wherein I-type collagen content highest in collagen in 29 had now been found that, accounts for about 90%.It is organized newly Form occur and cell metabolism in, assign new tissue mechanical intensity and biochemical characteristic；It is distributed mainly on skin, cornea, flesh The positions such as tendon play an important role to the normal physiological function and injury repair of maintenance cell, tissue etc..

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.

Though the correlative study of existing recombined human I-type collagen domestic at present, as Northwest University and South China Science & Engineering University are equal Research report in relation to I-type collagen small peptide, but it mainly studies collagen short peptide stretch, there is no related recombination human source The report of 1 chain full-length peptide of type i collagen α.According to current research achievement it is known that recombinant natural collagen short peptide stretch is degradable more Short small peptide, and because of its structural similarity, it is difficult to detect its purity.And 1 chain full-length peptide of recombination human source type i collagen α, more because of peptide chain It is long, it is easier to degrade, it more difficult to guarantee whether the collagen in product is full-length peptide, limits its extensive use.

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 because it is with higher molecular weight Unique physio-biochemical characteristics, as it is easily formed, one layer of ventilative film, anti-degradation time are long, are crosslinked post-crosslinking Du Genggao, property It can be more excellent；And its slow degradation for occurring over time, replenishing collagen small peptide that can be more longlasting, in addition with it is certain Drug combination can play slow releasing function.Therefore, in order to sufficiently develop its potential value, the production of its full-length peptide of urgent need to resolve with The test problems of overall length peptide chain.

Collagen isolates and purifies at present, mostly uses the 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；And the higher product of purity can be obtained because of its specific recognition in affinity purification technology, and easy to operate, is expected to solve Its purity is difficult to improve and purifying technique is difficult to stable problem.

Summary of the invention

The purpose of the present invention is to provide 1 catenin of a primary yeast recombination human source type i collagen α, synthetic method and its application, To solve the problems of the prior art.

To achieve the above object, the invention provides the following technical scheme:

One primary yeast recombination human source type i collagen α, 1 catenin, the amino acid sequence of 1 catenin of recombination human source type i collagen α Column are as shown in SEQ ID NO.1；1 catenin of recombination human source type i collagen α includes Strep-Tag II sequence, the source of people of N-terminal 6 × His sequence of I-type collagen α 1 chain maturation peptide sequence and C-terminal makes it contain bispecific affinity purification label；Overall length For 1071 amino acid.

Optimally, the expressed sequence of 1 catenin of recombination human source type i collagen α is as shown in SEQ ID NO.2.

Optimally, the gene order of the 1 chain mature peptide of source of people I-type collagen α is through utilizing the usual password of host What son obtained after optimizing natural collagen protein gene order, gene order is as shown in SEQ ID NO.3.

Optimally, the synthetic method of 1 catenin of a primary yeast recombination human source type i collagen α, comprising the following steps:

1) gene order of 1 chain mature peptide of source of people I-type collagen α is synthesized；

2) the transformation pPIC9k s containing Strep-Tag II coded sequence is prepared；

3) pPIC9k s is transformed in double digestion, constructs the recombinant plasmid containing 1 catenin gene of recombination human source type i collagen α pPIC9k-colIα1；

4) recombinant plasmid pPIC9k-colI α 1 is linearized with restriction enzyme Sal I, and its electrotransformation is entered to finish red In yeast SMD1168 competent cell, with nutrient defect type mark and the high copy positive recombinant of G418 resistance marker screening, obtain To pichia yeast genetic engineering bacteria；

5) fermented and cultured of pichia yeast genetic engineering bacteria；

6) after fermentation, centrifugal purification obtains 1 catenin of recombination human source type i collagen α.

Optimally, comprising the following steps:

1) gene order of 1 chain mature peptide of source of people I-type collagen α is synthesized: mature in 1 chain of source of people I-type collagen α Under the amino acid sequence permanence condition of peptide, by the password that corresponding Pichia pastoris is of little use in 1 chain gene sequence of people's type i collagen α Son is optimized for Pichia pastoris preferences codon；The gene of 1 chain mature peptide of source of people I-type collagen α is synthesized by full genome again Sequence；

A) first using pPIC9k plasmid as template, PCR amplification, product length 394bp are carried out using primer P1, P2；Its The gene order of middle P1 is as shown in SEQ ID NO.4, and the gene order of P2 is as shown in SEQ ID NO.5；

B) EcoRI and BamHI double digestion is done to the PCR product of acquisition and pPIC9k plasmid respectively, and is connected through T4 DNA Under enzyme effect, 16 DEG C of connections overnight, obtain transformation pPIC9k s；

3) the recombinant plasmid pPIC9k-colI α 1 containing 1 catenin gene of recombination human source type i collagen α is constructed:

A) using the gene order of the 1 chain mature peptide of source of people I-type collagen α synthesized in step 1) as template, primer is utilized P3, P4 carry out PCR amplification, obtain the genetic fragment that length is 3194bp；The wherein gene order of P3 such as SEQ ID NO.6 institute Show, the gene order of P4 is as shown in SEQ ID NO.7；

B) ApaI and Not I double digestion is done to transformation pPIC9k s obtained in step 2), obtains carrier segments；

C) it using P5, P6 as primer, using itself as template, is mutually expanded, is obtained containing 6 × His coded sequence by PCR Genetic fragment, length 65bp；Wherein the gene order of P5 is as shown in SEQ ID NO.8, the gene order of P6 such as SEQ ID Shown in NO.9；

D) each segment in step a), step b), step c) is mixed, is connected using the seamless Cloning Kit of instant, 50 DEG C of connection 1h are obtained containing Strep-Tag II coded sequence, 1 chain maturation peptide gene sequence of source of people I-type collagen α, 6 The recombinant plasmid pPIC9k-colI α 1 of × His coded sequence；

4) recombinant plasmid pPIC9k-colI α 1 is linearized with restriction enzyme Sal I in 37 DEG C, and by its electrotransformation Enter in Pichia pastoris SMD1168 competent cell, it is positive with histidine defect phenotypic marker and the high copy of G418 resistance marker screening Recon obtains pichia yeast genetic engineering bacteria；

5) fermented and cultured of pichia yeast genetic engineering bacteria: the pichia yeast genetic engineering bacteria prepared in step 4) is inoculated with In YPD fluid nutrient medium, 30 DEG C, 220rpm be activated overnight culture, then transfer in BMGY culture medium, in 30 DEG C, 220rpm cultivates engineering bacteria 16-18h, until OD₆₀₀=2.0-6.0；1500g is centrifuged 5min at room temperature, collects thallus；It uses again Thallus is resuspended in the BMMY culture medium of 10mL, makes OD₆₀₀=1.0 or so, resulting bacterium solution is placed in the sterile triangular flask of 100mL, Continue shaken cultivation under 28 DEG C, 220rpm；Methanol is added into culture medium within every 24 hours carry out induction training to final concentration of 1% It supports；

6) after fermentation, fermentation liquid is taken to be centrifuged off yeast cells, obtains fermentation supernatant；Under the conditions of non denatured, Ni- is used NTA His Bands resin adsorption recombinant collagen, washes away impurity with cleaning solution, finally elutes recombinant collagen again, collects eluent； Eluent is through ultrafiltration system desalination, concentration；Again by concentrate Strep-Tactin resin adsorption recombinant collagen, cleaning solution is used Impurity is washed away, finally elutes recombinant collagen again, collects eluent；Eluent is through ultrafiltration system desalination, concentration, obtained concentration 1 catenin of recombination human source type i collagen α is made through vacuum freeze drying in liquid.

Optimally, the collagen gel dressing of 1 catenin of yeast recombination human source type i collagen α preparation, the collagen egg Using purified water as solvent, component further includes 1 catenin of recombination human source type i collagen α, gelling agent, moisturizer for white gel dressing；Institute Stating gelling agent is carbomer, and the moisturizer is glycerol and triethanolamine；The wherein quality proportioning of each component are as follows: recombination human source I 1 catenin 0.1-0.5% of Collagen Type VI α, glycerol 1-10%, carbomer 0.1-4%, triethanolamine 0.1-10%, remaining is purifying Water；The gel dressing prepared in the present invention can increase moisture content of skin, adjust skin ph and grease, recombination especially therein 1 catenin of source of people type i collagen α can form one layer of ventilative film in skin surface, obstruct external bacterium, prevent surface of a wound inflammation, And wound healing, rebuild skin barrier function.Suitable for skin barrier function damage caused by dry skin furfur and its Reparation and Laser wounds reparation of his skin injury etc..

Optimally, the Collagen dressing of 1 catenin of yeast recombination human source type i collagen α preparation, the collagen Sticking dressing is made of 1 catenin of recombination human source type i collagen α, moisturizer, medical thickener and non-woven fabrics base material；The moisturizer For glycerol, the medical thickener is xanthan gum；The wherein quality proportioning of each component are as follows: 1 catenin of recombination human source type i collagen α 0.1-0.5%, glycerol 1-10%, xanthan gum 0.1-2%, remaining is purified water；The Collagen dressing prepared in the present invention Skin microcirculation can be effectively improved, relatively closed moist environment is built, enhances metabolism and the regeneration function of skin.It is applicable in In the rear early stage pigmentation of the surface of a wound, early stage superficial scars reparation etc..

Optimally, the importing product for beauty of 1 catenin of yeast recombination human source type i collagen α preparation, it is described to lead Entering property product each raw material component is as follows: by percentage to the quality, recombination human source type i collagen α 1 catenin 0.1-0.5%, glycerol 1- 10%, Sodium Hyaluronate 0.1-0.5%, micromolecule hyaluronic acid sodium 0.01-0.05%, sodium chloride 0.9%, SymPeptide 0.005-0.02 ‰, remaining is purified water；The importing product prepared in the present invention can to deep skin replenishing collagen, It can promote skin metabolism and full skin mitigate wrinkle, make the tender brilliant white high resilience of skin water.Suitable for liking to be beautiful, focusing on The crowd of skin care.

Optimally, the external-use skin care Essence of 1 catenin of yeast recombination human source type i collagen α preparation, the facial treatment essence Liquid each raw material component is as follows: by percentage to the quality, glycerol 1-3%, butanediol 1-3%, Sodium Hyaluronate 0.01-0.1%, again Group 1 catenin 0.01-0.1% of source of people I Collagen Type VI α, Vitamin C Ethyl Ether 0.01-0.1%, sodium ascorbyl phosphate 0.01- 0.1%, dipotassium glycyrrhizinate 0.1-0.5%, EDTANa₂0.01-0.1%, silk peptide 0.01-0.1%, beta glucan 0.1- 0.5%, reed fragrant plant oil 0.1-0.5%, water-solubleazone 0.1-1%, remaining is purified water；The external-use skin care essence prepared in the present invention Magnificent liquid can supplement skin-nourishing, improve skin microcirculation, and having keeps skin smooth wet and Bearberry Extract, and can obstruct external The intrusion of bacterium, dust, ultraviolet light protects the skin from infringement；Daily reparation suitable for skin is maintained.

The present invention logs in during optimizing the gene order of 1 chain mature peptide of source of people I-type collagen α according to Genbank Amino acid sequence and gene order, optimize its correspondence under the premise of not changing people's I-type collagen original amino acid Gene order, optimization processing is carried out according to Pichia pastoris preferences codon, and 1 chain mature peptide of people's type i collagen α is corresponding Gene order (colI α 1) in Pichia pastoris codon agg, cgt, ggg, ggc, gca etc. for being of little use to be optimized for its preference close Numeral aga, ggt, gct etc., by this processing, are more conducive to collagen using what is obtained after the analysis of DNASTAR software Expression；Simultaneously in the synthesis process, by the gene order of optimization eliminate ApaI, Bgl II, BamH I, EcoR I, Not I, The restriction endonuclease sites such as Sac I, Sal I.

In the transformation pPIC9k s being prepared in the present invention, Strep-Tag II coded sequence is next to plasmid On KEX2 protease site gene order after, improve the cutting efficiency of signal peptide, ensure that Strep-Tag II is not embedded and the high efficiency identification in purifying.

The recombinant yeast pichia pastoris genetic engineering bacterium prepared in the present invention has been preserved in Chinese microorganism strain preservation management committee Member's meeting common micro-organisms center, deposit number CGMCC No.17150, preservation date: on January 10th, 2019, address: Beijing No. 3 Institute of Microorganism, Academia Sinica, institute of Chaoyang District North Star West Road 1, classification naming: pichia pastoris yeast Pichia pastoris。

1 catenin of recombination human source type i collagen α being prepared in the present invention, chemical structure and performance are better than animal sources Collagen and prokaryotic micro-organisms source recombined collagen and recombinant collagen segment and gelatin in the market.

The present invention constructs eukaryotic expression system using Pichia pastoris, expresses recombination human source type i collagen α 1 by methanol induction Catenin, inducible promoter starting efficiency is high, and expression quantity is high；Fermentation period is short, and fermentation process is simple, is suitable for large-scale production It is produced with high density fermentation；Collagen is secreted extracellularly, and is easily isolated purifying；Product endotoxin-free, it is safer.

1 catenin of a primary yeast recombination human source type i collagen α is prepared in the present invention, overall length is 1071 amino acid, by N 6 × His sequence composition of the Strep-Tag II sequence at end, 1 chain maturation peptide sequence of source of people I-type collagen α and C-terminal, makes it It is marked containing bispecific affinity purification；The albumen is single-stranded structure；It is marked using the bispecific affinity purification that it has, easily In acquisition high purity product；The bispecific affinity purification being had using it is marked, convenient for detection and identification its product whether For overall length collagen.

1 catenin of yeast recombination human source type i collagen α that the present invention is produced using genetic recombination engineering bacteria has carried out opposite The purification process answered；In process of production, pass through the side of Ni-NTA HisBind resin and Strep-Tactin resin adsorption Formula extracts 1 catenin of recombination human source type i collagen α from the fermentation liquid of engineering bacteria and purifies it.

The codon that the present invention is had a preference for according to Pichia pastoris for the first time, the source of people I-type collagen α of artificial synthesized optimization 1 chain maturation peptide gene sequence constructs one and completely new, secreting type finishes red ferment by the methods of PCR amplification, digestion, connection Female efficient expression vector is sieved after foreign gene is imported Pichia pastoris by nutrient defect type mark screening, resistance The methods of choosing, expression screening, obtain the Pichia yeast engineering for capableing of 1 catenin of efficiently expressing recombinant human source type i collagen α Strain；Fermented and cultured, affinitive layer purification through specified conditions etc. operation, obtain nontoxic, high-purity product, can be used for dressing, The fields such as cosmetics, bio-medical raw material, shaping and beauty.

Compared with prior art, the beneficial effects of the present invention are:

1,1 catenin of recombination human source type i collagen α of the invention carries bispecific label at its two sections, can both lead to It crosses affinity purification and obtains overall length collagen, by its state in the product of sandwich method ELISA test sensitivity and can also contain Amount.

2, the gene order of 1 catenin of recombination human source type i collagen α of the invention is according to the usual codon of Pichia pastoris to it Gene order has carried out codon optimization, eliminates be of little use codon and hairpin structure, by synonymous conversion, optimizes mRNA's Secondary structure, avoid codon utilization efficiency limit caused by translation efficiency it is low, so that it is more suitable for the table in Pichia pastoris It reaches.

3, compared with conventional recombinant collagen segment and gelatin, 1 catenin of recombination human source type i collagen α is the collagen of overall length Albumen, thus there is very high molecular weight as large biological molecule and possess more preferably chemical structure and performance, it can be used as biology Medical material.

4, compared with traditional collagen production method, 1 catenin of recombination human source type i collagen α is eukaryotic expression system table The recombined collagen of the humanization reached, and can be secreted extracellularly, virus-free, endotoxin hidden danger, and avoid immune Rejection, biocompatibility and biological safety are higher, and for technical process to more environment-friendly, product is safer.

5, the hydrophily and stability of 1 catenin of recombination human source type i collagen α of the invention are good, amino acid composition and day Right collagen, amino acid sequence corresponding portion 100% is identical, and structure and bioactivity are closer to natural collagen protein.

6,1 catenin of recombination human source type i collagen α of the invention carry N-terminal Strep-Tag II label and C-terminal 6 × his label, using its bispecific affinity purification label, purification step is simple, and product purity is high, can meet minimally invasive, shaping The products such as beauty, bio-medical material use.

Detailed description of the invention

In order that the present invention can be more clearly and readily understood, right below according to specific embodiment and in conjunction with attached drawing The present invention is described in further detail.

Fig. 1 is the figure of the secreting, expressing recombinant plasmid pPIC9k-colI α 1 of 1 catenin of recombination human source type i collagen α of building Spectrum；

Fig. 2 is the PCR identification electrophoretogram for recombinating pPIC9k-colI α 1, and wherein swimming lane M is molecular weight Marker, 1-2 For different recombinant clones；

Fig. 3 is the SDS-PAGE of 1 catenin of recombinant yeast pichia pastoris engineering bacterium expression recombination human source type i collagen α screened Figure, wherein swimming lane M is molecular weight Marker, and 1-4 is to take the electrophoresis of 16 μ L of supernatant for 24 hours after different recombinant bacterial strain inducing expressions Figure；

Fig. 4 is the immunoblot experiment of recombination engineering expression 1 catenin of recombination human source type i collagen α screened, wherein M is molecular weight Marker, and 1,2 be the immunoblotting of same recombinant bacterial strain supernatant difference applied sample amount；3,4 be same recombinant bacterial strain The immunoblotting of supernatant difference applied sample amount；

Fig. 5 is the mass spectrum comparison result of recombinant bacterium expression 1 catenin of recombination human source type i collagen α screened；It is used in figure ProtTech ' s ProtQuest software suite software, according to UniProt protein database and NCBI number It is compared according to library；

Fig. 6 is that the overall length that sandwich method ELISA expresses 1 catenin of recombination human source type i collagen α to the recombinant bacterium screened is examined Test is fixed, and wherein degradation product is the protein degradation containing the end C label of purifying；Target protein is the target protein of purifying.

Specific embodiment

The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.

It is obvious to a person skilled in the art that the present invention is not limited to the details of following exemplary embodiment, Er Qie In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims Variation is included within the present invention.

Embodiment 1:

The preparation of 1 catenin of recombination human source type i collagen α is carried out in embodiment 1, experimental procedure is as follows:

1, it synthesizes the gene order of 1 chain mature peptide of source of people I-type collagen α: changing 1 chain of source of people I-type collagen α Under the conditions of the amino acid sequence of mature peptide, by the codon optimization that corresponding Pichia pastoris is of little use in its corresponding gene order For Pichia pastoris preferences codon, using DNASTAR software optimization gene order, and ApaI, Bgl II, BamH are eliminated I, the restriction endonuclease sites such as EcoR I, Not I, Sac I, Sal I, it is mature that synthesis obtains 1 chain of source of people I-type collagen α The gene order of peptide is shown in SEQ ID NO.3；

2, the transformation pPIC9k s containing Strep-Tag II coded sequence is prepared: first using pPIC9k plasmid as template, PCR amplification, product length 394bp are carried out using primer P1, P2；Wherein the gene order of P1 is as shown in SEQ ID NO.4, P2 Gene order as shown in SEQ ID NO.5；EcoRI and BamHI are done respectively to the PCR product of acquisition and pPIC9k plasmid again Double digestion, and through under the effect of T4 DNA ligase, 16 DEG C of connections are overnight；Connection product conversion is entered into competent E.coli again DH5 α, in the LB resistant panel screening positive clone containing Kan；Picking detects correct positive colony through PCR, mentions after culture Plasmid is taken, transformation pPIC9k s is obtained；

3, construct the recombinant plasmid pPIC9k-colI α 1 containing 1 catenin gene of recombination human source type i collagen α: first with The gene order of the 1 chain mature peptide of source of people I-type collagen α of synthesis is template, carries out PCR amplification using primer P3, P4, obtains Length is the genetic fragment of 3194bp；Wherein the gene order of P3 is as shown in SEQ ID NO.6, the gene order of P4 such as SEQ ID Shown in NO.7；ApaI and Not I double digestion is done to obtained transformation pPIC9k s again, obtains carrier segments；Then with P5, P6 is primer, using itself as template, is mutually expanded by PCR, obtains the genetic fragment containing 6 × His coded sequence, length For 65bp；Wherein the gene order of P5 is as shown in SEQ ID NO.8, and the gene order of P6 is as shown in SEQ ID NO.9；Finally will Each segment mixing of preparation, is connected, 50 DEG C of connection 1h using the seamless Cloning Kit of instant；Connection product is transformed into again Enter competent E.coli DH5 α, in the LB resistant panel screening positive clone containing Kan；Using primer P1, P7 to positive gram Grand progress PCR detection, wherein the gene order of P1 is as shown in SEQ ID NO.4, the gene order of P7 such as SEQ ID NO.10 institute Show, picking detects correct positive colony through PCR, extracts plasmid after culture, obtains containing Strep-Tag II coded sequence, people The recombinant plasmid pPIC9k-colI α 1 of 1 chain maturation peptide gene sequence of source I-type collagen α, 6 × His coded sequence；

4, the preparation of pichia yeast genetic engineering bacteria:

(1) linearisation of recombinant expression plasmid pPIC9k-colI α 1: recombinant plasmid pPIC9k-colI α 1 is extracted, with limitation Property restriction endonuclease Sal I is digested in 37 DEG C, then detects whether to cut completely through with 1% agarose gel electrophoresis, to complete After incision, digested liquid is handled with plastic recovery kit, recycles linearization plasmid, and desalting processing.

(2) preparation of 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. take the overnight culture of 50 μ L 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 OD₆₀₀Value reaches 1.1-1.3；

3. 1500 × g is centrifuged 5min by culture in 4 DEG C, thallus is resuspended with the aseptic double-distilled water that 50mL ice is pre-chilled；

4. being 3. centrifuged by step, thallus is resuspended with the aseptic double-distilled water that 25mL ice is pre-chilled；

5. being 3. centrifuged by step, thallus is resuspended in the sorbitol solution for the 1M being pre-chilled with 20mL ice；

6. being 3. centrifuged by step, 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, saved backup in -70 DEG C.

(3) electrotransformation of Pichia pastoris:

1. being rinsed electric revolving cup three times with dehydrated alcohol, residual ethanol is dried in sterilized super-clean bench；

2. electric revolving cup is sealed, goes to and 10min is pre-chilled in ice；

3. 1 matter of linearisation pPIC9k-colI α of about 10 μ g 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；25 μ F of capacitor；200 Ω of resistance；The electric shock time is 5-10mSec；

5. electric shock terminates, it 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 is coated on MD plate, every 100 μ L-200 μ L is coated with one flat plate, 10min is stored at room temperature, in 30 DEG C it is inverted culture 2-5 days, up to there is single colonie appearance.

(4) screening of multicopy 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 Gently scrape the His of planar surface⁺Transformant, and be transferred in 50mL centrifuge tube；

2. the dilution of 20mL aseptic double-distilled water is added, its OD is measured after mixing₆₀₀It is worth (1 OD₆₀₀=5 × 10⁷cells/mL)；

3. taking 10⁵A cell is coated on the YPD plate containing 0.5mg/mL G418, is inverted, 30 DEG C of culture 3-4d；

4. 200 μ LYPD fluid nutrient mediums are added in every hole in sterile 96 orifice plate；

5. being 4. respectively connected to toward step the conversion obtained on the YPD plate containing 0.5mg/mL G418 with sterile toothpick Son mixes, in 30 DEG C of culture 48h；

6. after 48h, taking one piece of new sterile 96 orifice plate, 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 taking one piece of new sterile 96 orifice plate, 190 μ L YPD fluid nutrient mediums are added in every hole；In corresponding aperture 10 μ L, second piece of 96 resulting culture of orifice plate is added, for 24 hours in 30 DEG C of cultures；

8. after for 24 hours, 1 μ L being taken out from 96 orifice plate of third block respectively and is put respectively and is containing 1.0mg/mL and 4mg/mL G418 YPD plate on, continue in 30 DEG C cultivate 96h-120h.

5, the pichia yeast genetic engineering bacteria of preparation the fermented and cultured of pichia yeast genetic engineering bacteria: is inoculated in YPD liquid In body culture medium, 30 DEG C, 220rpm be activated overnight culture, then transfer in BMGY culture medium, cultivate the work in 30 DEG C, 220rpm Journey bacterium 16-18h, until OD₆₀₀=2.0-6.0；1500g is centrifuged 5min at room temperature, collects thallus；The BMMY culture medium of 10mL is used again Thallus is resuspended, makes OD₆₀₀=1.0 or so, resulting bacterium solution is placed in the sterile triangular flask of 100mL, under 28 DEG C, 220rpm after Persistent oscillation culture；Methanol is added within every 24 hours into culture medium to final concentration of 1% progress Fiber differentiation；

6, it after fermentation, isolates and purifies, obtains recombined collagen:

(1) culture Pichia yeast engineering is activated overnight based on 30 DEG C, 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, 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；

(6) pure water of 5-7 times of volume is added in supernatant, then is concentrated by ultrafiltration to the 20% of initial volume；

(7) it takes and 100 μ L 1 × Ni-NTA combination buffers is added in 1mL concentrate, 4 DEG C are sufficiently mixed；

(8) 20 μ L 50%Ni-NTA HisBands resin suspensions are added softly to mix, in conjunction with 30min；

(9) 15000 × g are centrifuged 10 seconds precipitated resins, abandon supernatant；

(10) resin is rinsed with 100 μ L 1 × Ni-NTA wash buffers, 15000 × g is centrifuged 10 seconds, carefully sucks Clearly, it repeats primary；

(11) destination protein is eluted with 200 μ L 1 × Ni-NTA elution buffers, 15000 × g is centrifuged 10 seconds, carefully will Supernatant is transferred in clean tubule, is repeated 2 times；

(12) supernatant is collected, 4 DEG C of dialysed overnights of Strep combination buffer are used；

(13) it takes above-mentioned dialyzate that 100 μ L Strep-Tactin resin suspensions are added softly to mix, in conjunction with 30min；

(14) 15000 × g are centrifuged 10 seconds precipitated resins, abandon supernatant；

(15) with 200 μ L 1 × Strep-Tactin wash buffers rinse resin, 15000 × g be centrifuged 10 seconds, carefully Supernatant is sucked, is repeated primary；

(16) destination protein being eluted with 200 μ L 1 × Strep-Tactin elution buffers, 15000 × g is centrifuged 10 seconds, Carefully supernatant is transferred in clean tubule, is repeated 2 times；

(17) by obtained eluent through desalination, concentration, yeast weight is made through vacuum freeze drying in obtained concentrate Group collagen；

(18) take concentrate to pour into glass culture dish, be put into freeze overnight in -20 DEG C of refrigerators, be then transferred to it is pre- be cooled to - In 45 DEG C of freeze drier, vacuum pump is opened, maintains 48h；

(14) after being lyophilized, vent valve is carefully opened, until inside and outside air pressure balance；Culture dish is taken out, white ferment is obtained Female 1 catenin solid of recombination human source type i collagen α.

Experiment 1:

In above-mentioned steps 3, PCR detection, analytical electrophoresis institute are carried out by primer of P1/P7 to the positive colony that screening obtains Whether consistent with theoretical value 3685bp obtain stripe size；Experimental result is as shown in Figure 2.

Experiment 2:

Above-mentioned steps 6 the step of in (5), a small amount of supernatant is taken to carry out SDS-PAGE analysis detection, experimental result is as schemed Shown in 3.

Experiment 3:

Identification and sequencing are carried out to 1 catenin of recombination human source type i collagen α being prepared:

1, protein immunoblot (Western blotting), its step are as follows: fermented supernatant fluid being taken to carry out SDS-PAGE Electrophoresis, electrophoresis is until bromophenol blue is run out of from separation gel lower edge completely；Gel is taken out from glass plate, is soaked in transferring film buffering In liquid；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 and continues to soak Bubble is stand-by；In order transfer press from both sides in place impregnated in advance through transferring film buffer sponge, filter paper, pvdf membrane, gel, filter paper, Sponge guarantees do not have bubble between every layer；Transfer is folded up in electric turn trough, close to anode, glue turns film close to cathode, and by electricity Slot is placed in ice-water bath, and 450mA shifts 2h；Pvdf membrane is taken out, 5% skimmed milk power prepared with TBS solution is immersed in In, room temperature shakes 2h；Milk powder solution is discarded, the primary antibody prepared with 5% milk powder solution is added, room temperature shakes 2h；Primary antibody solution is poured out, with TBS solution washes film, washes three times, each 5min；Film is soaked into horseradish peroxidase (HRP) mark prepared with 5% milk powder solution In the goat anti-mouse IgG antibodies two corresponding anti-solution of note, room temperature shakes 2h；Two corresponding anti-solution is poured out, film is washed with TBS solution, is washed three times, often Secondary 5min；Residual moisture on film is blotted with filter paper, TMB developing solution (Beyotime) is uniformly coated on film, is placed in quiet at half-light Set a moment；Residual moisture on film, preservation of taking pictures are absorbed with filter paper；Testing result is as shown in Fig. 4.

2, mass spectral analysis identifies that its step are as follows: first taking protein sample to carry out SDS-PAGE electrophoretic analysis, uses coomassie Brilliant blue R-250 dyeing after decoloration, is moved towards to cut target stripe, sample presentation with 1mm width along target protein band with clean blade It is identified by general safe biology；Testing result is as shown in Figure 5.

Experiment 4:

Overall length is carried out to 1 catenin of recombination human source type i collagen α being prepared and examines measurement: utilizing yeast recombined human The parents and purification tag that the 1 catenin both ends source type i collagen α carry, can use Strep tag II monoclonal antibody and idol The 6xHis monoclonal antibody for joining HRP carries out sandwich method ELISA detection, with the overall length weight in qualitative or/and quantitative analysis sample Group 1 catenin of source of people type i collagen α.

Experimental method is as follows:

1) the anti-Strep tag II monoclonal antibody of mouse is diluted 1000 with combination buffer (1 × PBS buffer solution) Times, the antibody diluent in 100 holes μ L/ is added into elisa 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 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 of 100 μ L is added in every hole Dilution；

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, carry out the overall length yeast in sample survey 1 catenin of recombination human source I collagen α.

Testing result is as shown in fig. 6, its correspondence experimental data is as follows.

Embodiment 2:

Now provide embodiment of 1 catenin of yeast recombination human source type i collagen α for external application beauty skin care product:

By following quality proportioning, dissolve each raw material by solvent of purified water, and stir a colourless nothing The transparent preserving moisture and protecting skin Essence of taste；

Wherein glycerol 2%, butanediol 2%, Sodium Hyaluronate 0.5%, 1 catenin 0.5% of recombination human source I Collagen Type VI α, Vitamin C Ethyl Ether 0.5%, sodium ascorbyl phosphate 0.5%, dipotassium glycyrrhizinate 0.3%, EDTANa₂0.5%, silk peptide 0.5%, β-glucan 0.3%, reed fragrant plant oil 0.3%, water-solubleazone 0.5%, remaining is purified water.

Application method: after morning and evening face cleaning, being applied directly to face, gently pats to fully absorbing.

Embodiment 3:

Now provide embodiment of 1 catenin of recombination human source type i collagen α for external application beauty skin care product:

Wherein glycerol 1%, butanediol 1%, Sodium Hyaluronate 0.01%, 1 catenin of recombination human source I Collagen Type VI α 0.01%, Vitamin C Ethyl Ether 0.01%, sodium ascorbyl phosphate 0.01%, dipotassium glycyrrhizinate 0.1%, EDTA Na₂0.01%, silk peptide 0.01%, beta glucan 0.1%, reed fragrant plant oil 0.1%, water-solubleazone 0.1%, remaining is purified water.

Embodiment 4:

Wherein glycerol 3%, butanediol 3%, Sodium Hyaluronate 0.1%, 1 catenin 0.1% of recombination human source I Collagen Type VI α, Vitamin C Ethyl Ether 0.1%, sodium ascorbyl phosphate 0.1%, dipotassium glycyrrhizinate 0.5%, EDTANa₂0.1%, silk peptide 0.1%, β-glucan 0.5%, reed fragrant plant oil 0.5%, water-solubleazone 1%, remaining is purified water.

Embodiment 5:

Now provide Application Example of 1 catenin of yeast recombination human source type i collagen α in high-grade minimally invasive cosmetics:

For the importing product Micron Technology needle of high-grade minimally invasive beauty, solution is led to by dissolving each raw material as solvent using purified water It crosses aseptic procedure to be prepared, the steps include:

1. accurately weighing each component according to following quality proportioning, and mixing, constant volume is sufficiently stirred；Wherein yeast recombined human 1 catenin 0.3% of source type i collagen α, glycerol 5%, Sodium Hyaluronate 0.3%, micromolecule hyaluronic acid sodium 0.03%, sodium chloride 0.9%, SymPeptide 0.01 ‰.

2. with 0.22 μm of filtering with microporous membrane degerming；

3. aseptic subpackaged under aseptic condition, every filling 3mL.

4. being used in use, directly taking out cooperation and importing instrument.

Embodiment 6:

Now provide the application implementation that 1 catenin of yeast recombination human source type i collagen α prepares collagen gel casting product Example:

For the collagen gel using purified water as solvent, component further includes 1 chain egg of yeast recombination human source type i collagen α White, gelling agent, moisturizer；The gelling agent is carbomer；The moisturizer is glycerol and triethanolamine；

The quality proportioning of each component are as follows: 1 catenin 0.1% of yeast recombination human source type i collagen α, glycerol 1%, carbomer 0.1%, triethanolamine 0.1%, remaining is purified water.

Preparation step is as follows:

1. accurately weighing glycerol and carbomer, 1h is stirred, is sufficiently dissolved；

2. purified water is added, and 1 catenin of yeast recombination human source type i collagen α is accurately weighed, stirs 1h, sufficiently dissolve；

3. triethanolamine is added, continues to stir 1h, mix well to get collagen gel casting product.

Embodiment 7:

The quality proportioning of each component are as follows: 1 catenin 0.5% of yeast recombination human source type i collagen α, glycerol 10%, carbomer 4%, triethanolamine 10%, remaining is purified water.

Preparation step is as follows:

Embodiment 8:

The quality proportioning of each component are as follows: 1 catenin 0.3% of yeast recombination human source type i collagen α, glycerol 5%, carbomer 2%, triethanolamine 5%, remaining is purified water.

Preparation step is as follows:

Embodiment 9:

Now provide the Application Example that 1 catenin of yeast recombination human source type i collagen α prepares Collagen dressing product:

The Collagen dressing is by 1 catenin of yeast recombination human source type i collagen α, moisturizer, medical thickener and nothing Woven fabric substrate composition；The moisturizer is glycerol；The medical thickener is xanthan gum；

The wherein quality proportioning of each component are as follows: 1 catenin 0.1% of yeast recombination human source type i collagen α, glycerol 1%, xanthan Glue 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 treatment, irradiation sterilization is to get collagen Sticking dressing product.

Embodiment 10:

Now provide the application implementation that 1 catenin of yeast recombination human source type i collagen α prepares skin and mucosa protective agent casting product Example:

Skin and mucosa protective agent is made of the raw material of following quality proportioning: 1 catenin of yeast recombination human source type i collagen α 0.5%, Sodium Hyaluronate 0.5%, glycerol 20%, remaining is purified water；After obtained preparation solution sterilizing, sterile filling.

Preparation step is as follows:

1. accurately weighing each component, purified water dissolution is added, stirs 1h；

2. accurately weighing 1 catenin of yeast recombination human source type i collagen α, purified water is supplied, continues to stir, sufficiently dissolution is mixed It is even；

3. filling under aseptic condition, sealed package, irradiation sterilization to get collagen-base skin and mucosa protective agent dressing, Applied to the skin and mucosa protection etc. after micro-shaping.

Sequence table:

SEQUENCE LISTING

110 Jiangsu > Yue Zhi biological medicine Co., Ltd of <

120 > yeast recombination human source type i collagen α of <, 1 catenin

〈130〉UCSI1H

〈160〉10

〈170〉PatentIn version 3.5

The amino acid sequence of 1 catenin of SEQ ID NO.1 yeast recombination human source type i collagen α

WSHPQFEKQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPG EPGEPGASGPMGPR GPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGT AGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGEN GAPGQMGPRGLPGE RGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPR GSEGPQGV RGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPG ARGPSGPQGPGGPPGPKGNSGEPGAPGSKGD TGAKGEPGPVGVQGPPGPAGE EGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPA G PKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGP PGARGQAGVMGFPGPKGAAGEPG KAGERGVPGPPGAVGPAGKDGEAGAQG PPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLG APGPS GARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAP GLQGMPGERGAAGLPGPK GDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPA GAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPP GADGQPGAKG EPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGA AGRVGPPGPSGN AGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAG EKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQ RGERGFPGLPGPSGEPGK QGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDR GETG PAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAG PQGPRGDKGETGEQGDRGIKGHRGFSG LQGPPGPPGSPGEQGPSGASGPAGPR GPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPG PPSA GFDFSFLPQPPQEKAHDGGRYYRAHHHHHH

The gene DNA sequence of 1 catenin of SEQ ID NO.2 yeast recombination human source type i collagen α

TGGTCTCATCCACAATTTGAAAAGCAACTTAGTTATGGATACGATGAAAAAT CCACAGGTGGAATCA GTGTTCCTGGACCTATGGGTCCATCAGGTCCAAGAG GTTTACCAGGACCTCCAGGTGCCCCAGGTCCCCAGGGAT TTCAAGGTCCAC CAGGAGAGCCTGGTGAGCCAGGAGCTTCTGGTCCAATGGGTCCCAGAGGA CCACCTGGTCCT CCAGGAAAGAATGGAGATGATGGTGAAGCTGGAAAACC TGGAAGACCTGGAGAAAGAGGACCACCAGGACCCCAG GGTGCCAGAGGA CTGCCAGGTACCGCAGGTCTGCCTGGAATGAAAGGTCATAGAGGATTTTCA GGATTAGACGG TGCAAAGGGAGACGCTGGACCTGCAGGACCAAAGGGTG AGCCAGGAAGTCCAGGAGAGAATGGTGCACCAGGACA GATGGGTCCAAG AGGACTGCCCGGTGAAAGAGGTAGACCCGGAGCACCAGGACCAGCAGGT GCAAGAGGAAATG ATGGAGCTACAGGTGCTGCAGGACCCCCAGGTCCAAC AGGACCAGCCGGTCCTCCCGGTTTCCCAGGTGCCGTTG GAGCAAAAGGTG AAGCTGGTCCACAGGGTCCAAGAGGTTCTGAAGGTCCACAGGGAGTTAGA GGAGAACCAGGA CCCCCTGGACCAGCTGGTGCAGCAGGACCAGCTGGTA ACCCTGGTGCTGACGGTCAGCCAGGTGCTAAGGGAGCA AATGGAGCACCA GGAATAGCTGGTGCCCCAGGATTTCCCGGTGCTAGAGGTCCAAGTGGTCCA CAAGGACCAGG AGGTCCACCCGGTCCCAAAGGAAACAGTGGAGAACCAG GTGCACCCGGTTCAAAGGGAGATACAGGAGCTAAAGG AGAGCCCGGTCCA GTGGGTGTTCAGGGACCACCCGGACCTGCTGGAGAGGAAGGTAAAAGAG GTGCAAGAGGTG AGCCAGGACCAACAGGTCTGCCTGGTCCCCCTGGTGAA AGAGGTGGTCCAGGTAGTAGAGGATTTCCAGGAGCTG ATGGTGTTGCAGG ACCAAAGGGACCCGCAGGTGAGAGAGGATCACCCGGTCCAGCCGGACCA AAAGGATCACCA GGAGAAGCTGGTAGACCAGGAGAAGCTGGTCTGCCAG GTGCTAAAGGATTGACAGGATCACCCGGTTCACCTGGT CCTGATGGAAAGA CAGGACCTCCAGGTCCCGCTGGTCAGGACGGTAGACCAGGACCCCCAGGA CCCCCAGGTGC AAGAGGTCAGGCAGGTGTAATGGGTTTCCCCGGACCTAA AGGAGCAGCTGGAGAACCTGGTAAAGCTGGAGAGAG AGGAGTGCCTGGA CCCCCTGGAGCTGTTGGTCCAGCAGGAAAGGATGGTGAGGCAGGTGCACA AGGTCCACCTG GACCCGCTGGACCTGCAGGTGAGAGAGGAGAGCAAGGT CCCGCAGGTTCTCCAGGTTTTCAGGGTTTGCCAGGTC CAGCCGGTCCTCCT GGAGAGGCAGGAAAGCCAGGAGAACAAGGAGTTCCAGGAGACCTGGGTG CACCAGGACCC TCTGGTGCAAGAGGAGAGAGAGGATTTCCTGGAGAAAG AGGTGTGCAGGGACCACCAGGTCCCGCCGGTCCAAGA GGAGCAAATGGA GCCCCTGGAAATGACGGAGCTAAGGGTGACGCTGGTGCACCAGGAGCACC AGGTTCTCAAGG TGCTCCCGGATTGCAGGGTATGCCTGGAGAGAGAGGTG CAGCTGGACTGCCAGGTCCAAAAGGTGACAGAGGAGA CGCCGGTCCTAA GGGAGCTGACGGTTCTCCTGGAAAGGACGGTGTGAGAGGTTTGACAGGAC CAATAGGTCCAC CCGGTCCTGCTGGAGCCCCTGGAGACAAAGGTGAATCA GGTCCTTCCGGTCCAGCCGGACCAACAGGAGCAAGAG GAGCACCTGGAG ACAGAGGAGAGCCAGGTCCTCCAGGACCTGCAGGTTTCGCTGGTCCTCCC GGAGCAGATGGA CAGCCAGGAGCTAAGGGAGAACCCGGTGACGCTGGTG CTAAGGGAGATGCAGGTCCACCAGGTCCTGCTGGTCCT GCTGGACCTCCCG GACCAATAGGTAATGTTGGAGCACCCGGAGCAAAAGGTGCCAGAGGTTCC GCAGGTCCTCC CGGAGCAACTGGTTTTCCAGGAGCTGCCGGAAGAGTGGG TCCACCTGGTCCTTCTGGAAATGCAGGACCACCAGG TCCTCCTGGTCCAGC CGGAAAGGAAGGTGGAAAGGGACCTAGAGGAGAAACAGGTCCCGCAGGT AGACCCGGTG AGGTGGGTCCACCTGGTCCACCCGGTCCAGCTGGTGAGAA AGGAAGTCCTGGAGCAGACGGACCAGCTGGTGCCC CTGGTACACCAGGAC CCCAAGGAATAGCTGGTCAAAGAGGTGTTGTTGGTTTACCAGGTCAGAGA GGAGAAAGA GGTTTTCCAGGATTACCAGGTCCCTCAGGTGAGCCCGGAAA ACAGGGTCCCTCAGGAGCAAGTGGTGAAAGAGGA CCACCAGGACCAATG GGACCTCCAGGATTAGCTGGTCCACCAGGAGAATCAGGAAGAGAGGGTGC TCCTGGAGC AGAAGGTTCACCAGGAAGAGACGGTTCACCCGGAGCCAAG GGAGACAGAGGTGAAACAGGTCCCGCAGGTCCACC AGGAGCACCCGGAG CCCCTGGTGCTCCAGGACCTGTCGGACCAGCAGGAAAATCCGGTGACAGA GGTGAGACTG GACCCGCAGGTCCTGCTGGTCCTGTTGGACCAGTGGGTGC AAGAGGACCAGCAGGTCCACAAGGTCCAAGAGGTG ACAAAGGTGAGACA GGTGAGCAGGGTGACAGAGGAATTAAAGGTCACAGAGGATTTTCAGGACT GCAGGGACCA CCCGGTCCTCCCGGTTCCCCAGGAGAGCAAGGTCCATCCG GTGCATCCGGTCCAGCTGGACCCAGAGGACCACCT GGTTCTGCTGGTGCA CCAGGTAAAGATGGATTGAACGGTTTGCCTGGTCCAATAGGACCTCCTGGT CCAAGAGG AAGAACTGGTGACGCCGGTCCCGTCGGACCACCCGGTCCACC AGGTCCCCCAGGTCCACCCGGACCACCATCCGC AGGATTTGATTTCTCATT CCTTCCTCAACCTCCTCAAGAGAAAGCACATGATGGAGGTAGATACTATAG AGCCCATCACCACCATCATCATTAA

The DNA sequence dna of 1 chain mature peptide of SEQ ID NO.3 source of people I-type collagen α

CAACTTAGTTATGGATACGATGAAAAATCCACAGGTGGAATCAGTGTTCCT GGACCTATGGGTCCAT CAGGTCCAAGAGGTTTACCAGGACCTCCAGGTGCC CCAGGTCCCCAGGGATTTCAAGGTCCACCAGGAGAGCCTG GTGAGCCAGG AGCTTCTGGTCCAATGGGTCCCAGAGGACCACCTGGTCCTCCAGGAAAGA ATGGAGATGATGGT GAAGCTGGAAAACCTGGAAGACCTGGAGAAAGAGG ACCACCAGGACCCCAGGGTGCCAGAGGACTGCCAGGTACC GCAGGTCTGC CTGGAATGAAAGGTCATAGAGGATTTTCAGGATTAGACGGTGCAAAGGGA GACGCTGGACCTGC AGGACCAAAGGGTGAGCCAGGAAGTCCAGGAGAGA ATGGTGCACCAGGACAGATGGGTCCAAGAGGACTGCCCGG TGAAAGAGGT AGACCCGGAGCACCAGGACCAGCAGGTGCAAGAGGAAATGATGGAGCTA CAGGTGCTGCAGGAC CCCCAGGTCCAACAGGACCAGCCGGTCCTCCCGGT TTCCCAGGTGCCGTTGGAGCAAAAGGTGAAGCTGGTCCAC AGGGTCCAAG AGGTTCTGAAGGTCCACAGGGAGTTAGAGGAGAACCAGGACCCCCTGGAC CAGCTGGTGCAGCA GGACCAGCTGGTAACCCTGGTGCTGACGGTCAGCCA GGTGCTAAGGGAGCAAATGGAGCACCAGGAATAGCTGGT GCCCCAGGATT TCCCGGTGCTAGAGGTCCAAGTGGTCCACAAGGACCAGGAGGTCCACCCG GTCCCAAAGGAAA CAGTGGAGAACCAGGTGCACCCGGTTCAAAGGGAGA TACAGGAGCTAAAGGAGAGCCCGGTCCAGTGGGTGTTCA GGGACCACCCG GACCTGCTGGAGAGGAAGGTAAAAGAGGTGCAAGAGGTGAGCCAGGACC AACAGGTCTGCCTG GTCCCCCTGGTGAAAGAGGTGGTCCAGGTAGTAGAG GATTTCCAGGAGCTGATGGTGTTGCAGGACCAAAGGGAC CCGCAGGTGAG AGAGGATCACCCGGTCCAGCCGGACCAAAAGGATCACCAGGAGAAGCTG GTAGACCAGGAGAA GCTGGTCTGCCAGGTGCTAAAGGATTGACAGGATCA CCCGGTTCACCTGGTCCTGATGGAAAGACAGGACCTCCA GGTCCCGCTGG TCAGGACGGTAGACCAGGACCCCCAGGACCCCCAGGTGCAAGAGGTCAG GCAGGTGTAATGGG TTTCCCCGGACCTAAAGGAGCAGCTGGAGAACCTGG TAAAGCTGGAGAGAGAGGAGTGCCTGGACCCCCTGGAGC TGTTGGTCCAG CAGGAAAGGATGGTGAGGCAGGTGCACAAGGTCCACCTGGACCCGCTGG ACCTGCAGGTGAGA GAGGAGAGCAAGGTCCCGCAGGTTCTCCAGGTTTTC AGGGTTTGCCAGGTCCAGCCGGTCCTCCTGGAGAGGCAG GAAAGCCAGG AGAACAAGGAGTTCCAGGAGACCTGGGTGCACCAGGACCCTCTGGTGCA AGAGGAGAGAGAGGA TTTCCTGGAGAAAGAGGTGTGCAGGGACCACCAG GTCCCGCCGGTCCAAGAGGAGCAAATGGAGCCCCTGGAAAT GACGGAGCT AAGGGTGACGCTGGTGCACCAGGAGCACCAGGTTCTCAAGGTGCTCCCGG ATTGCAGGGTATGCC TGGAGAGAGAGGTGCAGCTGGACTGCCAGGTCCAA AAGGTGACAGAGGAGACGCCGGTCCTAAGGGAGCTGACGG TTCTCCTGGA AAGGACGGTGTGAGAGGTTTGACAGGACCAATAGGTCCACCCGGTCCTGC TGGAGCCCCTGGAG ACAAAGGTGAATCAGGTCCTTCCGGTCCAGCCGGAC CAACAGGAGCAAGAGGAGCACCTGGAGACAGAGGAGAGC CAGGTCCTCC AGGACCTGCAGGTTTCGCTGGTCCTCCCGGAGCAGATGGACAGCCAGGAG CTAAGGGAGAACCC GGTGACGCTGGTGCTAAGGGAGATGCAGGTCCACCA GGTCCTGCTGGTCCTGCTGGACCTCCCGGACCAATAGGT AATGTTGGAGCA CCCGGAGCAAAAGGTGCCAGAGGTTCCGCAGGTCCTCCCGGAGCAACTGG TTTTCCAGGAGC TGCCGGAAGAGTGGGTCCACCTGGTCCTTCTGGAAATGC AGGACCACCAGGTCCTCCTGGTCCAGCCGGAAAGGA AGGTGGAAAGGGA CCTAGAGGAGAAACAGGTCCCGCAGGTAGACCCGGTGAGGTGGGTCCACC TGGTCCACCCG GTCCAGCTGGTGAGAAAGGAAGTCCTGGAGCAGACGGA CCAGCTGGTGCCCCTGGTACACCAGGACCCCAAGGAA TAGCTGGTCAAAG AGGTGTTGTTGGTTTACCAGGTCAGAGAGGAGAAAGAGGTTTTCCAGGAT TACCAGGTCCC TCAGGTGAGCCCGGAAAACAGGGTCCCTCAGGAGCAAGT GGTGAAAGAGGACCACCAGGACCAATGGGACCTCCA GGATTAGCTGGTCC ACCAGGAGAATCAGGAAGAGAGGGTGCTCCTGGAGCAGAAGGTTCACCA GGAAGAGACGG TTCACCCGGAGCCAAGGGAGACAGAGGTGAAACAGGTC CCGCAGGTCCACCAGGAGCACCCGGAGCCCCTGGTGC TCCAGGACCTGTC GGACCAGCAGGAAAATCCGGTGACAGAGGTGAGACTGGACCCGCAGGTC CTGCTGGTCCTG TTGGACCAGTGGGTGCAAGAGGACCAGCAGGTCCACAA GGTCCAAGAGGTGACAAAGGTGAGACAGGTGAGCAGG GTGACAGAGGAA TTAAAGGTCACAGAGGATTTTCAGGACTGCAGGGACCACCCGGTCCTCCC GGTTCCCCAGGA GAGCAAGGTCCATCCGGTGCATCCGGTCCAGCTGGACC CAGAGGACCACCTGGTTCTGCTGGTGCACCAGGTAAA GATGGATTGAACG GTTTGCCTGGTCCAATAGGACCTCCTGGTCCAAGAGGAAGAACTGGTGAC GCCGGTCCCGT CGGACCACCCGGTCCACCAGGTCCCCCAGGTCCACCCGG ACCACCATCCGCAGGATTTGATTTCTCATTCCTTCC TCAACCTCCTCAAGAG AAAGCACATGATGGAGGTAGATACTATAGAGCC

SEQ ID NO.4 primers DNA sequences P1

GACTGGTTCCAATTGACAAGC

SEQ ID NO.5 primers DNA sequences P2

AGGGAATTCTACGTAGGGCCCCTTTTCAAATTGTGGATGAGACCATCTTTTC TCGAGAGATACCCC

SEQ ID NO.6 primers DNA sequences P3

GGTCTCATCCACAATTTGAAAAGCAACTTAGTTATGGATACGATGA

SEQ ID NO.7 primers DNA sequences P4

GGCTCTATAGTATCTACCTC

SEQ ID NO.8 primers DNA sequences P5

GGAGGTAGATACTATAGAGCCCATCACCACCATCATCATTAAC

SEQ ID NO.9 primers DNA sequences P6

AATTAATTCGCGGCCGCCCTAGGTTAATGATGATGGTG

SEQ ID NO.10 primers DNA sequences P7

GCAAATGGCATTCTGACATCC

Claims

1. 1 catenin of a primary yeast recombination human source type i collagen α, it is characterised in that: 1 catenin of recombination human source type i collagen α Amino acid sequence as shown in SEQ ID NO.1；1 catenin of recombination human source type i collagen α includes the Strep-Tag of N-terminal 6 × His sequence of II sequence, source of people I-type collagen α 1 chain maturation peptide sequence and C-terminal, makes it contain bispecific affine pure Change label；Overall length is 1071 amino acid.

2. 1 catenin of yeast recombination human source type i collagen α according to claim 1, it is characterised in that: the recombination human source I The expressed sequence of 1 catenin of Collagen Type VI α is as shown in SEQ ID NO.2.

3. 1 catenin of yeast recombination human source type i collagen α according to claim 2, it is characterised in that: the source of people I type glue The gene order of former 1 chain mature peptide of protein alpha is after being optimized natural collagen protein gene order using the usual codon of host It obtains, gene order is as shown in SEQ ID NO.3.

4. the synthetic method of 1 catenin of a primary yeast recombination human source type i collagen α, it is characterised in that: the following steps are included:

4) recombinant plasmid pPIC9k-colI α 1 is linearized with restriction enzyme Sal I, and its electrotransformation is entered into Pichia pastoris In SMD1168 competent cell, with nutrient defect type mark and the high copy positive recombinant of G418 resistance marker screening, finished Red yeast gene engineering bacteria；

5) fermented and cultured of pichia yeast genetic engineering bacteria；

5. the synthetic method of 1 catenin of yeast recombination human source type i collagen α according to claim 4, it is characterised in that: packet Include following steps:

1) gene order of 1 chain mature peptide of source of people I-type collagen α is synthesized: in the ammonia of 1 chain mature peptide of source of people I-type collagen α Under base acid sequence permanence condition, by the codon optimization that corresponding Pichia pastoris is of little use in 1 chain gene sequence of people's type i collagen α For Pichia pastoris preferences codon；The gene order of 1 chain mature peptide of source of people I-type collagen α is synthesized by full genome again；

A) first using pPIC9k plasmid as template, PCR amplification, product length 394bp are carried out using primer P1, P2；Wherein P1 Gene order as shown in SEQ ID NO.4, the gene order of P2 is as shown in SEQ ID NO.5；

B) EcoRI and BamHI double digestion is done to the PCR product of acquisition and pPIC9k plasmid respectively, and connects enzyme effect through T4DNA Under, 16 DEG C of connections overnight, obtain transformation pPIC9k s；

A) using the gene order of the 1 chain mature peptide of source of people I-type collagen α synthesized in step 1) as template, primer P3, P4 are utilized PCR amplification is carried out, the genetic fragment that length is 3194bp is obtained；Wherein the gene order of P3 is as shown in SEQ ID NO.6, P4's Gene order is as shown in SEQ ID NO.7；

B) ApaI and NotI double digestion is done to transformation pPIC9k s obtained in step 2), obtains carrier segments；

C) it using P5, P6 as primer, using itself as template, is mutually expanded by PCR, obtains the gene containing 6 × His coded sequence Segment, length 65bp；Wherein the gene order of P5 is as shown in SEQ ID NO.8, the gene order of P6 such as SEQ ID NO.9 institute Show；

D) each segment in step a), step b), step c) is mixed, is connected using the seamless Cloning Kit of instant, 50 DEG C 1h is connected, is obtained containing Strep-Tag II coded sequence, 1 chain maturation peptide gene sequence of source of people I-type collagen α, 6 × His The recombinant plasmid pPIC9k-colI α 1 of coded sequence；

4) recombinant plasmid pPIC9k-colI α 1 is linearized with restriction enzyme Sal I in 37 DEG C, and its electrotransformation is entered complete In red yeast SMD1168 competent cell, with histidine defect phenotypic marker and the high copy positive restructuring of G418 resistance marker screening Son obtains pichia yeast genetic engineering bacteria；

5) fermented and cultured of pichia yeast genetic engineering bacteria: the pichia yeast genetic engineering bacteria prepared in step 4) is inoculated in In YPD fluid nutrient medium, 30 DEG C, 220rpm be activated overnight culture, then transfer in BMGY culture medium, trained in 30 DEG C, 220rpm Engineering bacteria 16-18h is supported, until OD₆₀₀=2.0-6.0；1500g is centrifuged 5min at room temperature, collects thallus；The BMMY of 10mL is used again Thallus is resuspended in culture medium, makes OD₆₀₀=1.0 or so, resulting bacterium solution is placed in the sterile triangular flask of 100mL, in 28 DEG C, Continue shaken cultivation under 220rpm；Methanol is added within every 24 hours into culture medium to final concentration of 1% progress Fiber differentiation；

6) after fermentation, fermentation liquid is taken to be centrifuged off yeast cells, obtains fermentation supernatant；Under the conditions of non denatured, Ni-NTA is used His Bands resin adsorption recombinant collagen, washes away impurity with cleaning solution, finally elutes recombinant collagen again, collects eluent；Elution Liquid is through ultrafiltration system desalination, concentration；Again by concentrate Strep-Tactin resin adsorption recombinant collagen, washed away with cleaning solution miscellaneous Matter finally elutes recombinant collagen again, collects eluent；Eluent is through ultrafiltration system desalination, concentration, and obtained concentrate is through true 1 catenin of recombination human source type i collagen α is made in vacuum freecing-dry.

6. the collagen gel dressing of 1 catenin of yeast recombination human source type i collagen α preparation, it is characterised in that: the collagen egg Using purified water as solvent, component further includes 1 catenin of recombination human source type i collagen α, gelling agent, moisturizer for white gel dressing；Institute Stating gelling agent is carbomer, and the moisturizer is glycerol and triethanolamine；The wherein quality proportioning of each component are as follows: recombination human source I 1 catenin 0.1-0.5% of Collagen Type VI α, glycerol 1-10%, carbomer 0.1-4%, triethanolamine 0.1-10%, remaining is purifying Water.

7. the Collagen dressing of 1 catenin of yeast recombination human source type i collagen α preparation, it is characterised in that: the collagen Sticking dressing is made of 1 catenin of recombination human source type i collagen α, moisturizer, medical thickener and non-woven fabrics base material；The moisturizer For glycerol, the medical thickener is xanthan gum；The wherein quality proportioning of each component are as follows: 1 catenin of recombination human source type i collagen α 0.1-0.5%, glycerol 1-10%, xanthan gum 0.1-2%, remaining is purified water.

8. the importing product for beauty of 1 catenin of yeast recombination human source type i collagen α preparation, it is characterised in that: described to lead Entering property product each raw material component is as follows: by percentage to the quality, recombination human source type i collagen α 1 catenin 0.1-0.5%, glycerol 1- 10%, Sodium Hyaluronate 0.1-0.5%, micromolecule hyaluronic acid sodium 0.01-0.05%, sodium chloride 0.9%, SymPeptide 0.005- 0.02 ‰, remaining is purified water.

9. the external-use skin care Essence of 1 catenin of yeast recombination human source type i collagen α preparation, it is characterised in that: the facial treatment essence Liquid each raw material component is as follows: by percentage to the quality, glycerol 1-3%, butanediol 1-3%, Sodium Hyaluronate 0.01-0.1%, again Group 1 catenin 0.01-0.1% of source of people type i collagen α, Vitamin C Ethyl Ether 0.01-0.1%, sodium ascorbyl phosphate 0.01- 0.1%, dipotassium glycyrrhizinate 0.1-0.5%, EDTANa₂0.01-0.1%, silk peptide 0.01-0.1%, beta glucan 0.1- 0.5%, reed fragrant plant oil 0.1-0.5%, water-solubleazone 0.1-1%, remaining is purified water.