CN103102407A

CN103102407A - Genetic recombinant human-like collagen

Info

Publication number: CN103102407A
Application number: CN2013100332996A
Authority: CN
Inventors: 梁亮
Original assignee: XI'AN ELAGEN BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: Jiangsu Chuangjian Medical Technology Co ltd
Priority date: 2013-01-29
Filing date: 2013-01-29
Publication date: 2013-05-15
Anticipated expiration: 2033-01-29
Also published as: CN103102407B

Abstract

The invention discloses humanized genetic recombinant human-like collagen produced by eukaryotic bacteria. The humanized genetic recombinant human-like collagen has amino acid with the total length of 474, and is formed by connecting two sections of completely-identical human III-type collagen sections in series; the end C is connected with six histidine residue groups serving as specificity affinity purification markers. The performance of the genetic recombination human-like collagen is superior to animal collagens and original nuclear engineering bacteria recombinant collagens; and with the specificity affinity purification markers, the high-purity product is easily acquired.

Description

Gene recombination human collagen

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of recombined collagen and production method thereof.

Background technology

Collagen protein is a kind of Basic knowledge of analytical reagents, is form and the structure of keeping skin and histoorgan, is also the important substance of repairing each damaged tissue.Mainly being present in skin, bone, cartilage, tooth, tendon, ligament and the blood vessel of humans and animals, is structural protein important in reticular tissue, plays the function that supports organ, protection body.Collagen protein plays an important role to normal physiological function and the injury repairing of safeguarding cell, tissue, organ.Collagen protein can contain the necessary nutrient of skin layer of collagen protein, make active reinforcement of collagen protein in skin, the integrity that keeps stratum corneum moisture and fibrous texture, improve the metabolism of skin cells living environment and promotion skin histology, increase circulation, reach the skin care delaying retrogradation, beauty treatment, wrinkle-chasing, the purpose of hair care.

The traditional method of producing collagen protein is to utilize acid, alkali or enzyme to process animal tissues's (pigskin, ox-hide, donkey hide, fish-skin/squama etc.), therefrom extracts collagen protein.Although these methods are with low cost, the rate of recovery is higher, the collagen protein of producing is the mixing collagen peptide section that molecular weight is very little and length does not wait, and is commonly called as gelatin, is difficult to play the effect of moisturizing, nourishing; Extraction process is simply extensive, and product purity is not good, and peculiar smell is often arranged; Animal-origin can't be broken away from Biosafety hidden danger, has limited greatly the widespread use of traditional collagen in the various products such as makeup, food, medicine; When extracting, processing produces flood tide waste water grievous injury environment; Technology door mulberry is low, and raw material sources are wayward, causes leather shoes milk, toxic capsule to walk crosswise, and food, drug safety hidden danger are serious.

In order to explain the series of problems of traditional animal collagen, many scholars take up Applied Biotechnology and produce recombinant collagen, and because of all facilities of microorganism culturing, utilizing recombinant microorganism to produce collagen becomes main flow gradually.Industrialization is produced, and use the intestinal bacteria high density fermentation as Fan Daidi and cultivate the recombination human collagen of producing, and successful Application arrives cosmetic field.But the bacterial expression system has the Biosafety problems such as intracellular toxin, pyrogen, makes the high enterprise of production, testing cost of product, and has hidden danger; The albumen of expressing exists in bacterial cell with the inclusion body form, and product purification is difficult, the rate of recovery is restricted; Prokaryotic expression system is more rudimentary in addition, can not complete the translation post-treatment of expression product and modify, and makes product not have biological activity.

Therefore, increasing scholar begins to utilize eukaryotic microorganisms Restruction collagen: Zhang Fenglong etc. to utilize synthetic almost completely humanized's the recombinant collagen of pichia spp, is applied to makeup.But its purification process has only adopted traditional nonspecific macromole isolation technique such as ultrafiltration, gel-filtration, ion-exchange, purity is not high enough, foreign protein and Partial digestion albumen can't be thorough degree remove, can not meet Wicresoft's beauty treatment, shaping and beauty to the requirements at the higher level of purity.

Summary of the invention

Technical problem to be solved by this invention is the deficiency for above-mentioned existing recombinant collagen and technology of preparing thereof, and a kind of recombinant collagen of full-length human of eucaryon engineering bacteria production is provided, and its performance is much better than animal collagen, prokaryotic micro-organisms recombinant collagen; With specificity affinity purification mark, be easy to obtain high purity product simultaneously.

474 amino acid of gene recombination human collagen total length are in series by two sections identical human III type collagen fragment, and C-terminal connects 6 histidine residues, and (SEQ ID NO.1) is as follows for its sequence:

AGNTGAPGSP　GVSGPKGDAG　QPGEKGSPGA　QGPPGAPGPL　GIAGITGARG　50 LAGPPGMPGP　RGSPGPQGVK　GESGKPGANG LSGERGPPGP　QGLPGLAGTA　100 GEPGRDGNPG　SDGLPGRDGS　PGGKGDRGEN　GSPGAPGAPG　HPGPPGPVGP　150 AGKSGDRGES　GPAGPAGAPG　PAGSRGAPGP QGPRGDKGET　GERGAAGIKG　200 HRGFPGNPGA　PGSPGPAGQQ　GAIGSPGPAE FTAGNTGAPG　SPGVSGPKGD　250 AGQPGEKGSP　GAQGPPGAPG　PLGIAGITGA RGLAGPPGMP　GPRGSPGPQG 300

VKGESGKPGA　NGLSGERGPP　GPQGLPGLAG　TAGEPGRDGN　PGSDGLPGRD　350 GSPGGKGDRG　ENGSPGAPGA　PGHPGPPGPV　GPAGKSGDRG　ESGPAGPAGA　400 PGPAGSRGAP　GPQGPRGDKG　ETGERGAAGI　KGHRGFPGNP　GAPGSPGPAG 　450 QQGAIGSPGP　ADHHHHHHTG　LARF 　　 474

Albumen is single-stranded structure, wherein 1～229 and 233～461 is identical III Collagen Type VI peptide section, there is EFT to connect between two sections human III type collagen segments, adopt DHHHHHHTGLARF to modify after 233～461 the former fragment of human III type glue protein, wherein 463～468 is affinity purification mark 6His.

Gene recombination engineering bacteria and the protein preparation method of producing above-mentioned albumen are comprised of following step:

(1), the structure of recombinant expression vector

Extract total RNA from people's fresh human placenta, reverse transcription is cDNA, according to GenBank III type human collagen COL3A1 gene order, design pcr amplification primer F(SEQ ID NO.2), R(SEQ ID NO.3):

F: GAATCTGTGAATCATGCCCTAC

R: GAAGTCATAATCTCATCGGTGTT

Take cDNA as masterplate pcr amplification COL3A1 gene, product length is 3295bp.Reclaim the specificity product and be connected in the pGM-T carrier, obtain plasmid pGM-T-COL3A1;

According to the method for multiplex PCR in " molecular cloning experiment guide ", take pGM-T-COL3A1 plasmid as template, use primers F 1, F2, R to carry out the multiplex PCR amplification, obtain the product C OL3 that product is 722bp, primer is:

（SEQ ID NO.4）F1:AGGCTGAAGCTGCGGGTAACACTG

（SEQ ID NO.5）F2:TCTCTCGAGAAAAGAGAGGCTGAAGCT

（SEQ ID NO.6）R: TTGAATTCTGCAGGTCCTGG

Product C OL3 and carrier pPIC9K are done respectively EcoR I, Xho I double digestion, and be connected to become pPIC9K-COL3 through the T4 DNA ligase;

Take plasmid pPIC9K-COL3 as template, the design primer, the COL3 that again increases adds the 6His mark simultaneously.Primer is:

（SEQ ID NO.7）F:CCCGAATTCACTGCGGGTAACACTGGTGC

（SEQ ID NO.8）R:AAAACCGGTGTGGTGGTGGTGGTGGTGATCTGCAGGTCCTGGACTG

Secondary amplified production COL3-6His and plasmid pPIC9K-COL3 are carried out respectively EcoR I, Age I double digestion, and be connected to become pPIC9K-COL3-COL3-6His through the T4 DNA ligase.The gene order of target protein is as follows:

1 GCGGGTAACA CTGGTGCTCC TGGCAGCCCT GGAGTGTCTG GACCAAAAGG

51 TGATGCTGGC CAACCAGGAG AGAAGGGATC GCCTGGTGCC CAGGGCCCAC

101 CAGGAGCTCC AGGCCCACTT GGGATTGCTG GGATCACTGG AGCACGGGGT

151 CTTGCAGGAC CACCAGGCAT GCCAGGTCCT AGGGGAAGCC CTGGCCCTCA

201 GGGTGTCAAG GGTGAAAGTG GGAAACCAGG AGCTAACGGT CTCAGTGGAG

251 AACGTGGTCC CCCTGGACCC CAGGGTCTTC CTGGTCTGGC TGGTACAGCT

301 GGTGAACCTG GAAGAGATGG AAACCCTGGA TCAGATGGTC TTCCAGGCCG

351 AGATGGATCT CCTGGTGGCA AGGGTGATCG TGGTGAAAAT GGCTCTCCTG

401 GTGCCCCTGG CGCTCCTGGT CATCCAGGCC CACCTGGTCC TGTCGGTCCA

451 GCTGGAAAGA GTGGTGACAG AGGAGAAAGT GGCCCTGCTG GCCCTGCTGG

501 TGCTCCCGGT CCTGCTGGTT CCCGAGGTGC TCCTGGTCCT CAAGGCCCAC

551 GTGGTGACAA AGGTGAAACA GGTGAACGTG GAGCTGCTGG CATCAAAGGA

601 CATCGAGGAT TCCCTGGTAA TCCAGGTGCC CCAGGTTCTC CAGGCCCTGC

651 TGGTCAGCAG GGTGCAATCG GCAGTCCAGG ACCTGCAGAA TTCACTGCGG

701 GTAACACTGG TGCTCCTGGC AGCCCTGGAG TGTCTGGACC AAAAGGTGAT

751 GCTGGCCAAC CAGGAGAGAA GGGATCGCCT GGTGCCCAGG GCCCACCAGG

801 AGCTCCAGGC CCACTTGGGA TTGCTGGGAT CACTGGAGCA CGGGGTCTTG

851 CAGGACCACC AGGCATGCCA GGTCCTAGGG GAAGCCCTGG CCCTCAGGGT

901 GTCAAGGGTG AAAGTGGGAA ACCAGGAGCT AACGGTCTCA GTGGAGAACG

951 TGGTCCCCCT GGACCCCAGG GTCTTCCTGG TCTGGCTGGT ACAGCTGGTG

1001　AACCTGGAAG AGATGGAAAC CCTGGATCAG ATGGTCTTCC AGGCCGAGAT

1051 　GGATCTCCTG GTGGCAAGGG TGATCGTGGT GAAAATGGCT CTCCTGGTGC

1101 　CCCTGGCGCT CCTGGTCATC CAGGCCCACC TGGTCCTGTC GGTCCAGCTG

1151 　GAAAGAGTGG TGACAGAGGA GAAAGTGGCC CTGCTGGCCC TGCTGGTGCT

1201 　CCCGGTCCTG CTGGTTCCCG AGGTGCTCCT GGTCCTCAAG GCCCACGTGG

1251 　TGACAAAGGT GAAACAGGTG AACGTGGAGC TGCTGGCATC AAAGGACATC

1301 　GAGGATTCCC TGGTAATCCA GGTGCCCCAG GTTCTCCAGG CCCTGCTGGT

1351 　CAGCAGGGTG CAATCGGCAG TCCAGGACCT GCAGATCACC ACCACCACCA

1401 　CCACACCGGT　CTTGCTAGAT　TC　　　　　　　　　　　（SEQ ID NO.9）。

(2) structure of gene recombination engineering bacteria pichia spp

With the pPIC9K-COL3-COL3-6His restriction enzyme for preparing SalI linearizing, and preparation pichia spp SMD1168(Invitrogen) competent cell, electric shock transforms the pPIC9K-COL3-COL3-6His plasmid.Select high copy positive colony with the G418 gradient method, obtain pichia yeast genetic engineering bacteria and (be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC NO. 7189, preservation date: on January 21st, 2013, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Classification And Nomenclature: pasteur is than red yeast Pichia pastoris).

(3) preparation gene recombination human collagen

Cultivate based on 30 ℃ of 250rpm with BMGY and cultivated pichia yeast genetic engineering bacteria 16 ~ 18 hours, OD ₆₀₀=2～6; The centrifugal 5min of 1500g～3000g under room temperature collects thalline, with the resuspended thalline of BMMY substratum, makes OD ₆₀₀=1.0 left and right are placed in the bacterium liquid of gained the triangular flask of 500mL, with the double gauze sealing, in 30 ℃ of 250rpm continued growths; Adding methyl alcohol to final concentration in every 24 hours in the substratum is 1% beginning inducing culture, until more than 100 hours.After fermentation culture finishes, centrifugal collection supernatant, with Ni-NTA His Bands resin (Merck) absorption recombinant collagen, rinse streams goes out impurity under non-Denaturing, and wash-out recombinant collagen again, collect elutriant at last.Elutriant is through ultrafiltration system (PALL) desalination, concentrated.Concentrated solution obtained by freeze drying gene recombination human collagen.

As the functional raw material of makeup, the using method of gene recombination human collagen in makeup is described as follows:

Take the gene recombination human collagen lyophilized powder, add 5% N.F,USP MANNITOL in the 1mg:1mL ratio, fully after dissolving, filtration sterilization, under aseptic condition, every 1mL packing pours in the aseptic cillin bottle of 3mL, partly covers aseptic plug, after vacuum lyophilization, roll aluminium lid, make gene recombination human collagen and import lyophilized powder.Another preparation 0.05% hyaluronic acid solution, filtration sterilization is filled under aseptic condition in the aseptic cillin bottle of 3mL, covers aseptic plug, rolls aluminium lid, makes the lyophilized powder solvent.When the beauty salon uses, with the whole solvents of disposable sterilized injector extraction, inject the freeze-drying powder bottle, shake up and make abundant dissolving, and then the cooperation of extraction solution imports the instrument use.

The present invention compared with prior art has the following advantages:

1. traditional collagen of comparing, the immunological rejection that the gene recombination human collagen of full-length human has avoided animal collagen to bring has been stopped Biosafety hidden danger, and technological process is environmentally friendly;

2. the existing collagen from the protokaryon engineering bacteria of comparing, gene recombination human collagen is without intracellular toxin hidden danger;

3. the existing collagen from the eucaryon engineering bacteria of comparing, gene recombination human collagen carries specificity affinity purification mark, and purification step is simple, and product purity is very high, satisfies Wicresoft, the shaping and beauty product uses.

Description of drawings

Fig. 1 is carrier pPIC9K-COL3-COL3-6His constructing technology route map.

Fig. 2 is the electrophorogram of restructured Pichia pastoris in expression gene recombination human collagen.

Embodiment

Embodiment 1

474 amino acid of gene recombination human collagen total length are in series by two sections identical human III type collagen fragment, and end connects 6 histidine residues, and (SEQ ID NO.1) is as follows for its sequence:

The preparation method is as follows:

1. the structure of recombinant expression vector

1.1. RNA extracts (with reference to the TaKaRa RNAiso Plus of company reagent specification sheets)

(1) take people's fresh human placenta of freezing through very low temperature and organize approximately 50mg, be transferred to rapidly with in the mortar of Liquid nitrogen precooler, use pestle tissue abrasion, constantly add liquid nitrogen therebetween, until be ground into powder;

(2) add appropriate RNAiso Plus in mortar, pulverous sample is covered fully, then room temperature is standing, until sample melts fully, then continues to be ground to lysate with pestle and is transparence;

(3) homogenate is transferred in centrifuge tube standing 5 minutes of room temperature;

(4) centrifugal 5 minutes of 4 ℃ of 12,000g;

(5) draw supernatant liquor, move in new centrifuge tube;

(6) add chloroform (1/5 volume of RNAiso Plus), cover tightly the centrifuge tube lid, thermal agitation 15 seconds.After solution is fully emulsified, more standing 5 minutes of room temperature;

(7) centrifugal 15 minutes of 4 ℃ of 12,000g.Drawing supernatant liquor is transferred in another new centrifuge tube;

(8) add isopyknic Virahol, after the abundant mixing of the centrifuge tube that turns upside down, under 15～30 ℃ standing 10 minutes;

(9) centrifugal 10 minutes of 4 ℃ of 12,000g;

(10) careful supernatant discarded adds 75% ethanol lml lentamente along centrifugal tube wall, the washing centrifuge tube tube wall that turns upside down gently, and 4 ℃ of 12,000g carefully discard ethanol after centrifugal 5 minutes;

(11) the drying at room temperature precipitation is 2～5 minutes, adds appropriate RNase-free water dissolution precipitation, and dissolving is rear in-80 ℃ of preservations fully.

1.2. reverse transcription (with reference to the TaKaRa PrimeScript RT-PCR Kit of company specification sheets)

(1) the following mixed solution of preparation in the Microtube pipe:

Using amount of reagent

dNTP Mixture（10 mM each） 1μL

Random 6 mers（20 μM） 1μL

Template RNA 5μL

RNase Free dH2O supplies 10 μ L

(2) carry out sex change, annealing reaction on the PCR instrument: 65 ℃ of 5min, cooled on ice;

(3) the centrifugal several seconds makes the mixed solution of template ribonucleic acid/primer etc. be gathered in Microtube pipe bottom;

(4) the following inverse transcription reaction liquid of preparation in above-mentioned Microtube pipe:

Using amount of reagent

Above-mentioned sex change, annealing afterreaction liquid 10 μ L

5×PrimeScript Buffer 4μL

RNase Inhibitor（40 U/μL） 0.5μL

PrimeScript RTase 0.5 μL

RNase Free dH2O 5 μL

(5) carry out reverse transcription reaction by following condition on the PCR instrument: 30 ℃ of 10min, 42 ℃ of 30min, 70 ℃ of 10min, cooled on ice.

1.3. amplification COL3A1 gene (with reference to the TaKaRa PrimeScript RT-PCR Kit of company specification sheets)

According to GenBank III type human collagen COL3A1 gene order, design pcr amplification primer F, R:

F: GAATCTGTGA ATCATGCCCT AC （SEQ ID NO.2）

R: GAAGTCATAA TCTCATCGGT GTT （SEQ ID NO.3）

Prepare the PCR reaction solution by following composition:

Using amount of reagent

10×PCR Buffer Ⅱ 5μL

dNTP Mixture（10 mM each） 2μL

Primers F 0.5 μ L

Primer R 0.5 μ L

TaKaRa Ex Taq HS（5 U/μL） 0.5 μL

Above-mentioned inverse transcription reaction liquid≤5 μ L

RNase Free dH2O supplies 50 μ L

Reaction conditions is respectively: 95 ℃ of 5min---(94 ℃ of 30S---60 ℃ of 30S---72 ℃ of 2.5min) 4 ℃ of 10min of 30cycles---72 ℃ of 10min---.

1.4. preparation pGMT-COL3A1 plasmid

Utilize sepharose DNA to reclaim test kit (the TaKaRa Agarose Gel DNA Fragment Recovery Kit of company), press product description, reclaim the specific band COL3A1 of 3295bp from 1.5% common sepharose.By the biochemical pGM-T clone of day root test kit specification sheets, connect COL3A1 on the pGM-T carrier, transformed competence colibacillus E.coli DH 5 α, spread plate through blue hickie screening, is selected positive colony and is inoculated in 10mL LB substratum, and 37 ℃ of shaken overnight are cultivated.Obtain plasmid pGM-T-COL3A1.

1.5. double digestion COL3

(1) according to the method for multiplex PCR in " molecular cloning experiment guide ", take the pGM-T-COL3A1 plasmid as template, use primers F 1, F2, R to carry out the multiplex PCR amplification, obtain the product C OL3 that product is 722bp, primer is:

（SEQ ID NO.4）F1:AGGCTGAAGCTGCGGGTAACACTG

（SEQ ID NO.5）F2:TCTCTCGAGAAAAGAGAGGCTGAAGCT

（SEQ ID NO.6）R: TTGAATTCTGCAGGTCCTGG

Reaction system is:

Using amount of reagent

10×Reaction Buffer 5μL

dNTP Mixture (10 mM each) 2.0μL

pGM-T-COL3A1 5μL

Each 0.5 μ L of Primer F1/F2 (20 μ M)

Primer R (20μM) 1μL

Taq enzyme 0.5 μ L

ddH ₂O supplies 50 μ L

Reaction conditions is:

95℃ 5 min——（94℃ 30 S——60℃ 30 S——72℃ 40S）30 cycles－—72℃ 10 min

(2) utilize sepharose DNA to reclaim test kit (the TaKaRa Agarose Gel DNA Fragment Recovery Kit of company), press product description, reclaim the specific band COL3 of 722bp from 1.5% common sepharose;

(3) COL3 after the recovery carries out double digestion by 37 ℃ of water-baths digestion 1.5～2.0h of following system:

Using amount of reagent

10×buffer 20μL

COL3(150ng/μL) 40μL

EcoRⅠ 5μL

XhoⅠ 5μL

ddH ₂O supplies 200 μ L

First take out 5 μ L before termination reaction and detect with 0.7% agarose gel electrophoresis whether enzyme cuts entirely;

(4) utilize sepharose DNA to reclaim test kit (the TaKaRa Agarose Gel DNA Fragment Recovery Kit of company), press product description, reclaim enzyme slitting band from 1.5% common sepharose ,-20 ℃ of preservations are stand-by.

1.6. build the pPIC9K-COL3 plasmid

(1) get freezing DH5 α competent cell one pipe, be put on ice and thaw, add immediately plasmid pPIC9K (＜=50ng), finger flicks and makes its mixing, placed 30 minutes in ice;

Lucky 90 seconds of (2) 42 ℃ of water-bath heat shocks are transferred to pipe in ice bath fast, make cooling 1 – of cell 2 minutes;

(3) add the LB liquid nutrient medium of 800 μ L antibiotic-frees, mixing, 37 ℃ of incubations 50 minutes;

(4) draw 100-200 μ L bacterium liquid, be applied on the solid LB flat board that contains kantlex (50 μ g/mL);

(5) 37 ℃ of incubators are inverted and were cultivated 12-18 hour;

(6) select single colony inoculation in the liquid LB substratum that contains kantlex (50 μ g/mL), 37 ℃, 150rpm shaking table overnight incubation is utilized the plasmid extraction kit operation instructions afterwards, and the plasmid that carries out pPIC9K extracts in a large number;

(7) by a large amount of double digestion pPIC9K of following system:

Using amount of reagent

10×buffer 20μL

pPIC9K (200ng/μL) 35μL

XhoⅠ 5μL

EcoRⅠ 5μL

ddH ₂O supplies 200 μ L

(8) press glue and reclaim test kit (the TaKaRa Agarose Gel DNA Fragment Recovery Kit of company), product description reclaims enzyme slitting band;

(9) mix by following system, the COL3 of double digestion is connected with pPIC9K connects 2 hours under 16 ℃:

Using amount of reagent

COL3(100 ng/μL) 11μL

pPIC9K (200ng/μL) 5μL

buffer 2μL

T4 DNA ligase 2 μ L

(10) connect product by a large amount of pPIC9K-COL3 plasmids of above-mentioned steps (1)～(6) preparation ,-20 ℃ of preservations are stand-by.

1.7. preparation COL3-6His

(1) take plasmid pPIC9K-COL3 as template, the design primer, the COL3 that again increases adds the 6His mark simultaneously.Primer is:

F:CCCGAATTCACTGCGGGTAACACTGGTGC（SEQ ID NO.7）

R:AAAACCGGTGTGGTGGTGGTGGTGGTGATCTGCAGGTCCTGGACTG（SEQ ID NO.8）

Reaction system is:

Using amount of reagent

10×Reaction Buffer 5μL

dNTP Mixture (10 mM each) 1.0μL

pPIC9K-COL3 1μL

Primer F (20μM) 1μL

Primer R (20μM) 1μL

Taq enzyme 1 μ L

ddH ₂O supplies 50 μ L

Reaction conditions is:

94℃ 5 min——（94℃ 30 S——55℃ 30 S——72℃ 1min）30 cycles——72℃ 10 min

(2) utilize sepharose DNA to reclaim test kit (the TaKaRa Agarose Gel DNA Fragment Recovery Kit of company), press product description, reclaim specific band COL3-6His from sepharose.

1.8. preparation pPIC9K-COL3-COL3-6His plasmid

(1) COL3-6His and plasmid pPIC9K-COL3 are pressed respectively following system EcoRI and a large amount of double digestions of AgeI:

Using amount of reagent

10×buffer 20μL

COL3-6His or pPIC9K-COL3 (200ng/ μ L) 35 μ L

AgeI 5μL

EcoRⅠ 5μL

ddH ₂O supplies 200 μ L

Reclaim the purpose fragment, be connected to become the pPIC9K-COL3-COL3-6His plasmid by following system:

Using amount of reagent

pPICK9-COL3 0.5μL

COL3-6His 5μL

Buffer 1μL

T4 ligase enzyme 0.5 μ L

ddH ₂O supplies 10 μ L

(2) step by (1)～(6) in 1.6 prepares the pPIC9K-COL3-COL3-6His plasmid in a large number, and-20 ℃ of preservations are stand-by.

2. the structure of recombinant yeast pichia pastoris engineering bacteria

2.1. the linearizing of pPIC9K-COL3-COL3-6His plasmid

Get restriction enzyme SalI 10 μ L, about pPIC9K-COL3-COL3-6His plasmid 20 μ g, add 100 μ L buffer H, add water and supply mixing after 1000 μ L, 37 ℃ of water-bath digestion 5h first take out 5 μ L reaction solutions before termination reaction, detect with 0.7% agarose gel electrophoresis whether enzyme cuts entirely, enzyme carries out plasmid extraction with plasmid extraction kit to linearization plasmid after cutting entirely.

2.2. the preparation of pichia spp (SMD1168) competent cell

(1) picking yeast list bacterium colony is seeded to and contains in 5mL YPD substratum test tube, 30 ℃, 250rpm overnight incubation;

(2) culture of getting 50 μ L is seeded in the 300mL triangular flask that contains the 50mL fresh culture, and 28 ~ 30 ℃, 250rpm overnight incubation are to OD ₆₀₀Value reaches 1.1-1.3;

(3) with culture in 4 ℃, the centrifugal 5min of 1500g, with the sterilized water of the ice precooling of 50mL, that bacterial sediment is resuspended;

(4) centrifugal by step (3), the sterilized water of the ice precooling of use 25mL is resuspended with bacterial sediment;

(5) centrifugal by step (3), the Sorbitol Solution USP of the 1mol/L of the ice precooling of use 20mL is resuspended with bacterial sediment;

(6) centrifugal by step (3), the Sorbitol Solution USP of the 1mol/L of the ice precooling of use 0.3mL is resuspended with bacterial sediment, and its final volume is about 0.5mL;

(7) every 80 μ L packing are a ,-70 ℃ of preservations.

2.3. the electricity of pichia spp transforms

(1) rinse electricity with dehydrated alcohol and transform cup three times, dry residual ethanol in the super clean bench of the bacterium of going out;

(2) electricity is transformed cup and went in ice precooling 10 minutes;

(3) the linearizing pPIC9K-COL3-COL3-6His plasmid with 5 ~ 20 μ g is dissolved in 5 ~ 10 μ L sterilization distilled waters, and with pichia spp competent cell mixing, the electricity that goes to the precooling of 0.2cm ice transforms in cup;

(4) electric shock, voltage 1.5kV; Electric capacity 25 μ F; Resistance 200 Ω; The electric shock time is 4 ~ 10mSec;

(5) electric shock complete after, add the Sorbitol Solution USP of 1mol/L of 1mL ice precooling with the thalline mixing, go in the 1.5mL centrifuge tube;

(6) the thalline suspension is coated on the MD flat board every 100 μ L ~ 200 μ L coating one flat plates.

2.4. multiple copied inserts the screening of recon

(1) there is the MD planar surface of transformant to add 2mL sterilization distilled water in growth, then is coated with gently on His+ transformant surface with aseptic triangle spreader and scrapes, and transfer in the 50mL centrifuge tube;

(2) add the dilution of 20mL sterilization distilled water, measure its OD600 value (1 OD600=5 * 10 after mixing ⁷Cells/mL);

(3) get 10 ⁵Individual cell is coated on the YPD flat board that contains 0.5mg/mL G418, is inverted, and cultivates 72 ~ 96h for 30 ℃;

(4) every hole adds 200 μ L YPD liquid nutrient mediums in aseptic 96 orifice plates;

(5) access respectively the transformant that obtains on the YPD flat board of 0.5mg/mL G418 containing toward step (4) with aseptic toothpick, mixing is cultivated 48h in 30 ℃;

(6) after 48h, get new aseptic 96 orifice plates, every hole adds 190 μ L YPD liquid nutrient mediums.The culture that adds first 96 orifice plate gained of 10 μ L in respective aperture is cultivated 24h in 30 ℃;

(7) after 24h, then get new aseptic 96 orifice plates, every hole adds 190 μ L YPD liquid nutrient mediums.The culture that adds second 96 orifice plate gained of 10 μ L in respective aperture is cultivated 24h in 30 ℃;

(8) after 24h, take out respectively 1 μ L and put respectively on the YPD flat board that contains 1.0mg/mL and 4.0mg/mL G418 from the 3rd 96 orifice plates, continue to cultivate 96h-120h in 30 ℃.If yeast SMD1168 transformant can be grown containing on the substratum of high density G418 more, illustrate that this transformant contains the more goal gene of multiple copied, namely have a plurality of pPIC9K-COL3-COL3-6His segments transform into yeast SMD1168 and homologous recombination inserted on the karyomit(e) of yeast.

3. preparation gene recombination human collagen

(1) cultivated pichia yeast genetic engineering bacteria 16 ~ 18 hours based on 30 ℃/250rpm, OD with the BMGY cultivation ₆₀₀=2～6;

(2) the centrifugal 5min of 1500g～3000g under room temperature collects thalline, with the resuspended thalline of BMMY substratum, makes OD ₆₀₀=1.0 left and right are placed in gained bacterium liquid the triangular flask of 500mL, with the double gauze sealing, in 30 ℃ of 250rpm continued growths;

(3) adding methyl alcohol to final concentration in every 24 hours in the substratum is 1% beginning inducing culture, until more than 100 hours;

(4) after fermentation culture finished, 4 ℃ of centrifugal 20min of lower 3000g collected supernatant;

(5) add the pure water of 5～7 times of volumes in supernatant, then through ultrafiltration and concentration to 20% of original volume;

(6) get and add 100 μ L 1 * Ni-NTA binding buffer liquid in the 1mL concentrated solution, 4 ℃ are fully mixed;

(7) add 4 ℃ of soft mixings of 20 μ L 50% Ni – NTA HisBind resin suspensions, in conjunction with 30 minutes;

(8) the centrifugal 10s precipitation of 15,000g resins are abandoned supernatant;

(9) with 100 μ L 1 * Ni-NTA rinsing damping fluid rinsing resins, centrifugal 10 seconds of 15,000g carefully sucks supernatant.Repeat again 1 time;

(10) with 20 μ L 1 * Ni-NTA elution buffer wash-out target proteins, centrifugal 10 seconds of 15,000 g carefully are transferred to supernatant in clean tubule.Repeat again 3 times.The supernatant that takes a morsel carries out the SDS-PAGE analyzing and testing;

(11) collect supernatant, add the pure water of 5 times of volumes, then through ultrafiltration and concentration to 10～20% of original volume;

(12) get the 10ml concentrated solution and pour in the 50ml small beaker, put into-20 ℃ of refrigerator freezings and spend the night, then change in freeze drier-45 ℃ over to, open vacuum pump, kept 48 hours;

(13) after freeze-drying finishes, carefully open purging valve, until inside and outside air pressure balance.Take out beaker, obtain the white powder gene recombination human collagen.

Embodiment 2

Now provide the embodiment that gene recombination human collagen is used for external application beauty and skin care product:

By following quality proportioning, with each raw material of water dissolution, and stir the transparent preserving moisture and protecting skin essence of a colorless and odorless:

Gene recombination human collagen 1%

Hyaluronate sodium 0.1%

Beta-glucan 1%

Jojoba ester 0.5%

Serine 0.1%

Sorbyl alcohol 5%

Trimethyl-glycine 5%

Sodium.alpha.-hydroxypropionate 3%

Using method: sooner or later after clean, directly be applied in face, chucked is to absorbing fully.

Embodiment 3:

Gene recombination human collagen 0.01%

Hyaluronate sodium 0.01%

Beta-glucan 0.01%

Jojoba ester 0.01%

Serine 0.01%

Sorbyl alcohol 1%

Trimethyl-glycine 1%

Sodium.alpha.-hydroxypropionate 0.1%

Embodiment 4

Now provide the Application Example of gene recombination human collagen in high-grade Wicresoft cosmetics:

(1) accurately take gene recombination human collagen lyophilized powder 10.0mg, be dissolved in 5% mannitol solution, fully be settled to 10.0mL after dissolving;

(2) with 0.22 μ m micro emulsion membrane filtration degerming;

(3) under aseptic condition, every 1.0mL packing pours in the aseptic cillin bottle of 3mL, partly covers aseptic plug, after vacuum lyophilization, rolls aluminium lid, makes gene recombination human collagen and imports lyophilized powder;

(4) separately prepare 0.05% sodium hyaluronate solution, according to the method filtration sterilization of (2);

(5) be filled under aseptic condition in the aseptic cillin bottle of 3mL, every bottle of 3.0mL adds a cover aseptic plug, rolls aluminium lid, makes the lyophilized powder solvent;

When (6) using, with the whole solvents of disposable sterilized injector extraction, inject the freeze-drying powder bottle, shake up and make abundant dissolving, and then the cooperation of extraction solution imports the instrument use.

Embodiment 5

Now provide the acute oral toxicity test embodiment that gene recombination human collagen carries out as cosmetic material:

Laboratory animal: 20 healthy mices, 8～12 ages in week, male and female half and half, body weight 25～30 grams;

Raising condition: full nutrition granulated feed feeding, sub-cage rearing, 23 ± 3 ℃ of room temperatures, relative humidity 40 ~ 70%, illumination every day 12 hours, free tap water and searching for food.Before test, animal needs to adapt to 3 days in experimental situation;

Dosage: during prerun, do not find that sample 5000mg/kg body weight dosage has overt toxicity reaction and dead, therefore test is to greatest extent adopted once in formal experiment, dosage is made as the 5000mg/kg body weight;

Preparation: accurately take sample 3.0 grams, be dissolved in pure water, be settled to 30mL, be mixed with 10% concentration, as test liquid.

Testing sequence

Contaminating mode: give testing liquid by the amount of 0.2mL/10g body weight minute three per os gavages in 24 hours, front 12 hours of gavage is freely drunk water, and after gavage, fasting is 3～4 hours, but normal diet then;

Observe time limit and index: observed 14 days, and recorded animal toxicity symptom and death condition every day, in the observation period, surviving animals is weighed weekly, and after the observation period finishes, surviving animals is weighed, and LD is calculated in postmortem after putting to death ₅₀

Test result: after gavage, all animal subjects have no obvious toxicity symptom and death within the observation period.Therefore its mouse oral toxicity LD ₅₀The 5000mg/kg body weight.

Tested material is to its mouse oral toxicity test result

Dosage (mg/kg)	Laboratory animal number (only)	Dead animal number (only)	Mortality ratio
				5000	20	0	0.0

Conclusion: according to the acute oral toxicity evaluation regulation of " cosmetics health standard " (version in 2007), the nontoxic level in the true border of gene recombination human collagen sample.

Embodiment 6

Now provide the acute dermal toxicity experimental example that gene recombination human collagen carries out as cosmetic material:

Laboratory animal: 10 healthy rats, 8～12 ages in week, male and female half and half, body weight 200～250 grams;

Dosage: during prerun, do not find that sample 2500mg/kg body weight dosage has overt toxicity reaction and dead, therefore test is to greatest extent adopted once in formal experiment, dosage is made as the 2500mg/kg body weight;

Preparation: by every the weight of animals weighing given the test agent, add several pure water furnishing pasty states.

Testing sequence

Contaminating mode: tested front 24 hours, with the back part of animal unhairing, area is about 40cm ², given the test agent evenly is applied in the unhairing district, cover with gauze and oilpaper, then fix with medical proof fabric.Contaminate after 24 hours, remove, and clean residual sample on skin with warm water;

Test result: all animal subjects have no obvious toxicity symptom and death within the observation period.Therefore rat percutaneous toxicity LD ₅₀The 2500mg/kg body weight.

Tested material is to rat oral skin toxicity test result

Dosage (mg/kg)	Laboratory animal number (only)	Dead animal number (only)	Mortality ratio
				2500	10	0	0.0

Conclusion: according to the acute dermal toxicity evaluation regulation of " cosmetics health standard " (version in 2007), the nontoxic level in the true border of gene recombination human collagen sample.

SEQUENCE LISTING

＜110〉the glad bio tech ltd of Xi'an benefit power

＜120〉gene recombination human collagen

<130> ELG33H6

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 474

<212> PRT

＜213〉artificial sequence

<220>

<221> SIMILAR

<222> (1)..(229)

＜223〉human III type collagen segment

<220>

<221> CHAIN

<222> (1)..(474)

＜223〉gene recombination human collagen complete sequence

<220>

<221> SIMILAR

<222> (233)..(461)

＜223〉human III type collagen segment

<220>

<221> DOMAIN

<222> (462)..(474)

＜223〉the modification fragment of C-terminal

<220>

<221> DOMAIN

<222> (463)..(468)

＜223〉6His fragment

<400> 1

Ala Gly Asn Thr Gly Ala Pro Gly Ser Pro Gly Val Ser Gly Pro Lys

1 5 　　10 　15

Gly Asp Ala Gly Gln Pro Gly Glu Lys Gly Ser Pro Gly Ala Gln Gly

　　20 　　　25 　　　30

Pro Pro Gly Ala Pro Gly Pro Leu Gly Ile Ala Gly Ile Thr Gly Ala

　35 　　　40 　　　45

Arg Gly Leu Ala Gly Pro Pro Gly Met Pro Gly Pro Arg Gly Ser Pro

　50 　　　55 　　　60

Gly Pro Gln Gly Val Lys Gly Glu Ser Gly Lys Pro Gly Ala Asn Gly

65 　　　70 　　　75 　　　80

Leu Ser Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Leu Pro Gly Leu

　　　85 　　　90 　　　95

Ala Gly Thr Ala Gly Glu Pro Gly Arg Asp Gly Asn Pro Gly Ser Asp

　　100 　　　105 　　　110

Gly Leu Pro Gly Arg Asp Gly Ser Pro Gly Gly Lys Gly Asp Arg Gly

　　　115 　　　120 　　　125

Glu Asn Gly Ser Pro Gly Ala Pro Gly Ala Pro Gly His Pro Gly Pro

　130 　　　135 　　　140

Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Ser

145 　　　150 　　　155 　　　160

Gly Pro Ala Gly Pro Ala Gly Ala Pro Gly Pro Ala Gly Ser Arg Gly

　　　165 　　　170 　　　175

Ala Pro Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu

　　180 　　　185 　　　　190

Arg Gly Ala Ala Gly Ile Lys Gly His Arg Gly Phe Pro Gly Asn Pro

　　195 　　200 　　　205

Gly Ala Pro Gly Ser Pro Gly Pro Ala Gly Gln Gln Gly Ala Ile Gly

　210 　　　215 　　　220

Ser Pro Gly Pro Ala Glu Phe Thr Ala Gly Asn Thr Gly Ala Pro Gly

225 　　　230 　　　235 　　　240

Ser Pro Gly Val Ser Gly Pro Lys Gly Asp Ala Gly Gln Pro Gly Glu

　　　245 　　　250 　　　255

Lys Gly Ser Pro Gly Ala Gln Gly Pro Pro Gly Ala Pro Gly Pro Leu

　　260 　　　265 　　　270

Gly Ile Ala Gly Ile Thr Gly Ala Arg Gly Leu Ala Gly Pro Pro Gly

　275 　　280 　　　285

Met Pro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gly Val Lys Gly Glu

　290 　　　295 　　300

Ser Gly Lys Pro Gly Ala Asn Gly Leu Ser Gly Glu Arg Gly Pro Pro

305 　　　310 　　　315 　　　320

Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Ala Gly Glu Pro Gly

　　　325 　　　330 　　　335

Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly Arg Asp Gly Ser

　　340 　　　345 　　　350

Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Ser Pro Gly Ala Pro

　355 　　　360 　　　365

Gly Ala Pro Gly His Pro Gly Pro Pro Gly Pro Val Gly Pro Ala Gly

　370 　　　375 　　　380

Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro Ala Gly Ala

385 　　　390 　　　395 　　　400

Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pro Gln Gly Pro Arg

　　　405 　　　410 　　　415

Gly Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Ala Gly Ile Lys Gly

　　420 　　　425 　　　430

His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro

　435 　　　440 　　　445

Ala Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pro Ala Asp His His

　450 　　　455 　　460

His His His His Thr Gly Leu Ala Arg Phe

465 　　470

<210> 2

<211> 22

<212> DNA

<213> Homo sapiens

<400> 2

gaatctgtga atcatgccct ac 22

<210> 3

<211> 23

<212> DNA

<213> Homo sapiens

<400> 3

gaagtcataa tctcatcggt gtt 23

<210> 4

<211> 24

<212> DNA

<213> Homo sapiens

<400> 4

aggctgaagc tgcgggtaac actg 24

<210> 5

<211> 27

<212> DNA

<213> Homo sapiens

<400> 5

tctctcgaga aaagagaggc tgaagct 27

<210> 6

<211> 20

<212> DNA

<213> Homo sapiens

<400> 6

ttgaattctg caggtcctgg 20

<210> 7

<211> 29

<212> DNA

＜213〉artificial sequence

<400> 7

cccgaattca ctgcgggtaa cactggtgc 29

<210> 8

<211> 46

<212> DNA

＜213〉artificial sequence

<400> 8

aaaaccggtg tggtggtggt ggtggtgatc tgcaggtcct ggactg 46

<210> 9

<211> 1422

<212> DNA

＜213〉artificial sequence

<220>

<221> CDS

<222> (1)..(1422)

＜223〉gene recombination human collagen gene order

<400> 9

gcg ggt aac act ggt gct cct ggc agc cct gga gtg tct gga cca aaa 48

Ala Gly Asn Thr Gly Ala Pro Gly Ser Pro Gly Val Ser Gly Pro Lys

1 　　　5 　　　10 　　 15

ggt gat gct ggc caa cca gga gag aag gga tcg cct ggt gcc cag ggc 96

Gly Asp Ala Gly Gln Pro Gly Glu Lys Gly Ser Pro Gly Ala Gln Gly

　　 20 　　　25 　　　30

cca cca gga gct cca ggc cca ctt ggg att gct ggg atc act gga gca 144

Pro Pro Gly Ala Pro Gly Pro Leu Gly Ile Ala Gly Ile Thr Gly Ala

　 35 　　　40 　　45

cgg ggt ctt gca gga cca cca ggc atg cca ggt cct agg gga agc cct 192

Arg Gly Leu Ala Gly Pro Pro Gly Met Pro Gly Pro Arg Gly Ser Pro

　50 　　　55 　　　60

ggc cct cag ggt gtc aag ggt gaa agt ggg aaa cca gga gct aac ggt 240

Gly Pro Gln Gly Val Lys Gly Glu Ser Gly Lys Pro Gly Ala Asn Gly

65 　　　70 　　　75 　　　80

ctc agt gga gaa cgt ggt ccc cct gga ccc cag ggt ctt cct ggt ctg 288

Leu Ser Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Leu Pro Gly Leu

　　　85 　　　 90 　　　95

gct ggt aca gct ggt gaa cct gga aga gat gga aac cct gga tca gat 336

Ala Gly Thr Ala Gly Glu Pro Gly Arg Asp Gly Asn Pro Gly Ser Asp

　　100 　　　105 　　　110

ggt ctt cca ggc cga gat gga tct cct ggt ggc aag ggt gat cgt ggt 384

Gly Leu Pro Gly Arg Asp Gly Ser Pro Gly Gly Lys Gly Asp Arg Gly

　　115 　　　120 　　125

gaa aat ggc tct cct ggt gcc cct ggc gct cct ggt cat cca ggc cca 432

Glu Asn Gly Ser Pro Gly Ala Pro Gly Ala Pro Gly His Pro Gly Pro

　130 　　　135 　　　140

cct ggt cct gtc ggt cca gct gga aag agt ggt gac aga gga gaa agt 480

Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Ser

145 　　150 　　　155 　　　160

ggc cct gct ggc cct gct ggt gct ccc ggt cct gct ggt tcc cga ggt 528

Gly Pro Ala Gly Pro Ala Gly Ala Pro Gly Pro Ala Gly Ser Arg Gly

　　　165 　　170 　　　175

gct cct ggt cct caa ggc cca cgt ggt gac aaa ggt gaa aca ggt gaa 576

Ala Pro Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu

　　180 　　　185 　　　190

cgt gga gct gct ggc atc aaa gga cat cga gga ttc cct ggt aat cca 624

Arg Gly Ala Ala Gly Ile Lys Gly His Arg Gly Phe Pro Gly Asn Pro

　195 　　200 　　　205

ggt gcc cca ggt tct cca ggc cct gct ggt cag cag ggt gca atc ggc 672

Gly Ala Pro Gly Ser Pro Gly Pro Ala Gly Gln Gln Gly Ala Ile Gly

　210 　　215 　　　220

agt cca gga cct gca gaa ttc act gcg ggt aac act ggt gct cct ggc 720

Ser Pro Gly Pro Ala Glu Phe Thr Ala Gly Asn Thr Gly Ala Pro Gly

225 　　230 　　　235 　　240

agc cct gga gtg tct gga cca aaa ggt gat gct ggc caa cca gga gag 768

Ser Pro Gly Val Ser Gly Pro Lys Gly Asp Ala Gly Gln Pro Gly Glu

　　245 　　　250 　　255

aag gga tcg cct ggt gcc cag ggc cca cca gga gct cca ggc cca ctt 816

Lys Gly Ser Pro Gly Ala Gln Gly Pro Pro Gly Ala Pro Gly Pro Leu

　　260 　　265 　　　270

ggg att gct ggg atc act gga gca cgg ggt ctt gca gga cca cca ggc 864

Gly Ile Ala Gly Ile Thr Gly Ala Arg Gly Leu Ala Gly Pro Pro Gly

　275 　　280 　　　285

atg cca ggt cct agg gga agc cct ggc cct cag ggt gtc aag ggt gaa 912

Met Pro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gly Val Lys Gly Glu

　290 　　295 　　　300

agt ggg aaa cca gga gct aac ggt ctc agt gga gaa cgt ggt ccc cct 960

Ser Gly Lys Pro Gly Ala Asn Gly Leu Ser Gly Glu Arg Gly Pro Pro

305 　　　310 　　　315 　　　320

gga ccc cag ggt ctt cct ggt ctg gct ggt aca gct ggt gaa cct gga 1008

Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Ala Gly Glu Pro Gly

　　　325 　　　330 　　　335

aga gat gga aac cct gga tca gat ggt ctt cca ggc cga gat gga tct 1056

Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly Arg Asp Gly Ser

　　340 　　　345 　　　350

cct ggt ggc aag ggt gat cgt ggt gaa aat ggc tct cct ggt gcc cct 1104

Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Ser Pro Gly Ala Pro

　　355 　　　360 　　　365

ggc gct cct ggt cat cca ggc cca cct ggt cct gtc ggt cca gct gga 1152

Gly Ala Pro Gly His Pro Gly Pro Pro Gly Pro Val Gly Pro Ala Gly

　370 　　375 　　　380

aag agt ggt gac aga gga gaa agt ggc cct gct ggc cct gct ggt gct 1200

Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro Ala Gly Ala

385 　　　390 　　　395 　　　400

ccc ggt cct gct ggt tcc cga ggt gct cct ggt cct caa ggc cca cgt 1248

Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pro Gln Gly Pro Arg

　　　405 　　　410 　　　415

ggt gac aaa ggt gaa aca ggt gaa cgt gga gct gct ggc atc aaa gga 1296

Gly Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Ala Gly Ile Lys Gly

　　420 　　　425 　　　430

cat cga gga ttc cct ggt aat cca ggt gcc cca ggt tct cca ggc cct 1344

His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro

　　435 　　　440 　　　 445

gct ggt cag cag ggt gca atc ggc agt cca gga cct gca gat cac cac 1392

Ala Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pro Ala Asp His His

　450 　　　455 　　460

cac cac cac cac acc ggt ctt gct aga ttc 1422

His His His His Thr Gly Leu Ala Arg Phe

465 　　470

Claims

1. humanization gene recombination collagen protein, it is characterized in that sequence is as follows: 474 amino acid of albumen total length, wherein 1～229 and 233～461 is the identical former fragment of human III type glue protein, there is EFT to connect between two sections human III type collagen segments, adopts DHHHHHHTGLARF to modify after 233～461 the former fragment of human III type glue protein; Its aminoacid sequence is as follows: (SEQ ID NO.1)

VKGESGKPGA　NGLSGERGPP　GPQGLPGLAG　TAGEPGRDGN　PGSDGLPGRD　350 GSPGGKGDRG　ENGSPGAPGA　PGHPGPPGPV　GPAGKSGDRG　ESGPAGPAGA　400 PGPAGSRGAP　GPQGPRGDKG　ETGERGAAGI　KGHRGFPGNP　GAPGSPGPAG 　450 QQGAIGSPGP　ADHHHHHHTG　LARF　　　　　　　　　　　　　　　　　　　 474。

2. engineering bacteria of producing the described albumen of claim 1 is characterized in that this project bacterium is obtained by following step:

(1) clone's human III type glue protein gene

Extract total RNA from people's fresh human placenta, reverse transcription is cDNA, according to GenBank III type human collagen COL3A1 gene order, and design gene PCR amplimer F, R:

（SEQ ID NO.2）F: GAATCTGTGAATCATGCCCTAC

（SEQ ID NO.3）R: GAAGTCATAATCTCATCGGTGTT

Amplification obtains gene fragment take cDNA as masterplate, and product length is 3295bp;

(2) build plasmid pPIC9K-COL3-COL3-6His

Utilize sepharose DNA to reclaim test kit, press product description, reclaim specific band COL3A1 from 1.5% common sepharose; According to pGM-T clone test kit specification sheets, COL3A1 is connected on the pGM-T carrier, obtain plasmid pGM-T-COL3A1; Take the pGM-T-COL3A1 plasmid as template ,Increase as primer carries out multiplex PCR take F1, F2, R, obtaining product C OL3 length is 722bp, and primer is:

（SEQ ID NO.4）F1:AGGCTGAAGCTGCGGGTAACACTG

（SEQ ID NO.5）F2:TCTCTCGAGAAAAGAGAGGCTGAAGCT

（SEQ ID NO.6）R: TTGAATTCTGCAGGTCCTGG

Product C OL3 and plasmid pPIC9K are done respectively EcoR I, Xho I double digestion, and be connected to become plasmid pPIC9K-COL3 through the T4 DNA ligase;

Take plasmid pPIC9K-COL3 as template, the COL3 that again increases obtains COL3-6His, and primer is:

F:CCCGAATTCACTGCGGGTAACACTGGTGC （SEQ ID NO.7）

R:AAAACCGGTGTGGTGGTGGTGGTGGTGATCTGCAGGTCCTGGACTG（SEQ ID NO.8）

Secondary amplified production COL3-6His and plasmid pPIC9K-COL3 are carried out respectively EcoRI, AgeI double digestion, and be connected to become plasmid pPIC9K-COL3-COL3-6His through the T4 DNA ligase;

(3) pichia yeast genetic engineering bacteria of gene recombination construction expression human III type glue protein

With the plasmid pPIC9K-COL3-COL3-6His restriction enzyme for preparing SalI linearizing, and preparation pichia spp SMD1168(Invitrogen) competent cell, electric shock transforms the pPIC9K-COL3-COL3-6His plasmid, selects high copy positive colony with the G418 gradient method, obtains pichia yeast genetic engineering bacteria.

3. the method for preparing the described albumen of claim 1 is characterized in that cultivating based on 30 ℃ of 250rpm with BMGY and cultivated pichia yeast genetic engineering bacteria claimed in claim 2 16 ~ 18 hours, to OD ₆₀₀=2～6; The centrifugal 5min of 1500g～3000g under room temperature collects thalline, with the resuspended thalline of BMMY substratum, makes OD ₆₀₀=1.0 left and right are placed in triangular flask with the bacterium liquid of gained, with the double gauze sealing, in 30 ℃ of 250rpm continued growths; Adding methyl alcohol to final concentration in every 24 hours in the substratum is 1% beginning inducing culture, until more than 100 hours; After fermentation culture finishes, centrifugal collection supernatant, with Ni-NTA His Bands resin (Merck) absorption recombinant collagen, rinse streams goes out impurity under non-Denaturing, and wash-out recombinant collagen again, collect elutriant at last; Elutriant is through ultrafiltration system (PALL) desalination, concentrated, concentrated solution obtained by freeze drying gene recombination human collagen.

4. the gene order of the described albumen of claim 1 of encoding, is characterized in that sequence is as follows: (SEQ ID NO.9)

1 GCGGGTAACA CTGGTGCTCC TGGCAGCCCT GGAGTGTCTG GACCAAAAGG

51 TGATGCTGGC CAACCAGGAG AGAAGGGATC GCCTGGTGCC CAGGGCCCAC

101 CAGGAGCTCC AGGCCCACTT GGGATTGCTG GGATCACTGG AGCACGGGGT

151 CTTGCAGGAC CACCAGGCAT GCCAGGTCCT AGGGGAAGCC CTGGCCCTCA

201 GGGTGTCAAG GGTGAAAGTG GGAAACCAGG AGCTAACGGT CTCAGTGGAG

251 AACGTGGTCC CCCTGGACCC CAGGGTCTTC CTGGTCTGGC TGGTACAGCT

301 GGTGAACCTG GAAGAGATGG AAACCCTGGA TCAGATGGTC TTCCAGGCCG

351 AGATGGATCT CCTGGTGGCA AGGGTGATCG TGGTGAAAAT GGCTCTCCTG

401 GTGCCCCTGG CGCTCCTGGT CATCCAGGCC CACCTGGTCC TGTCGGTCCA

451 GCTGGAAAGA GTGGTGACAG AGGAGAAAGT GGCCCTGCTG GCCCTGCTGG

501 TGCTCCCGGT CCTGCTGGTT CCCGAGGTGC TCCTGGTCCT CAAGGCCCAC

551 GTGGTGACAA AGGTGAAACA GGTGAACGTG GAGCTGCTGG CATCAAAGGA

601 CATCGAGGAT TCCCTGGTAA TCCAGGTGCC CCAGGTTCTC CAGGCCCTGC

651 TGGTCAGCAG GGTGCAATCG GCAGTCCAGG ACCTGCAGAA TTCACTGCGG

701 GTAACACTGG TGCTCCTGGC AGCCCTGGAG TGTCTGGACC AAAAGGTGAT

751 GCTGGCCAAC CAGGAGAGAA GGGATCGCCT GGTGCCCAGG GCCCACCAGG

801 AGCTCCAGGC CCACTTGGGA TTGCTGGGAT CACTGGAGCA CGGGGTCTTG

851 CAGGACCACC AGGCATGCCA GGTCCTAGGG GAAGCCCTGG CCCTCAGGGT

901 GTCAAGGGTG AAAGTGGGAA ACCAGGAGCT AACGGTCTCA GTGGAGAACG

951 TGGTCCCCCT GGACCCCAGG GTCTTCCTGG TCTGGCTGGT ACAGCTGGTG

1001 AACCTGGAAG AGATGGAAAC CCTGGATCAG ATGGTCTTCC AGGCCGAGAT

1051 GGATCTCCTG GTGGCAAGGG TGATCGTGGT GAAAATGGCT CTCCTGGTGC

1101 CCCTGGCGCT CCTGGTCATC CAGGCCCACC TGGTCCTGTC GGTCCAGCTG

1151 GAAAGAGTGG TGACAGAGGA GAAAGTGGCC CTGCTGGCCC TGCTGGTGCT

1201 CCCGGTCCTG CTGGTTCCCG AGGTGCTCCT GGTCCTCAAG GCCCACGTGG

1251 TGACAAAGGT GAAACAGGTG AACGTGGAGC TGCTGGCATC AAAGGACATC

1301 GAGGATTCCC TGGTAATCCA GGTGCCCCAG GTTCTCCAGG CCCTGCTGGT

1351 CAGCAGGGTG CAATCGGCAG TCCAGGACCT GCAGATCACC ACCACCACCA

1401 CCACACCGGT CTTGCTAGAT TC。

5. recombined collagen purification process when be used for using gene recombination engineering bacteria Restruction people derived collagen albumen, it is characterized in that in carrying out gene recombination engineering bacteria building process, when carrying out recombination human source collagen, amino acid sequences Design, use is accompanied with the nucleotide primer of protein modification function, adopt the mode of additional DHHHHHHTGLARF to modify to people's derived collagen Argine Monohydrochloride sequence, and pass through the mode purifying of Ni – NTA HisBind resin absorption when extracting the recombination human source collagen protein from engineering bacterium fermentation liquid.

6. one kind is used for claimed in claim 5ly, is accompanied with the nucleotide primer of protein modification function, when it is characterized in that according to people's derived collagen Argine Monohydrochloride sequences Design nucleotide primer, increases by 6 nucleotide sequences that are used for expressing Histidine of design at the C end.

7. being used for according to claim 6 increasing by 6 nucleotide sequences that are used for the expression Histidine at the C end is GTGGTGGTGGTGGTGGTG.

8. the nucleotide primer that is accompanied with the protein modification function claimed in claim 6 is characterized in that its nucleotides sequence classifies as

F:CCCGAATTCACTGCGGGTAACACTGGTGC

R:AAAACCGGTGTGGTGGTGGTGGTGGTGATCTGCAGGTCCTGGACTG 。

9. gene recombination human collagen claimed in claim 1 is in the purposes of preparation in makeup.

10. an external-use skin care product that is used for beauty treatment, is characterized in that prepared product take water as solvent, is comprised of the raw material of following quality proportioning:

Gene recombination human collagen 0.01～1%

Hyaluronate sodium 0.01～0.1%

Beta-glucan 0.01～1%

Jojoba ester 0.01～0.5%

Serine 0.01～1%

Sorbyl alcohol 1～5%

Trimethyl-glycine 1～5%

Sodium.alpha.-hydroxypropionate 0.1～3%.

11. an importing product that is used for beauty treatment is characterized in that respectively by lyophilized powder and solvent composition, lyophilized powder is formed by the aseptic preparation of raw material of following quality proportioning, and lyophilize:

Gene recombination human collagen 0.1%

N.F,USP MANNITOL 5%;

Solvent is comprised of 0.05% aseptic sodium hyaluronate solution.