CN114195884B

CN114195884B - Recombinant human collagen and preparation method thereof

Info

Publication number: CN114195884B
Application number: CN202111264263.XA
Authority: CN
Inventors: 杨宇丰; 李钧翔; 陆益
Original assignee: Hemei Biotechnology Zhejiang Co ltd
Current assignee: Hemei Biotechnology Zhejiang Co ltd
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2024-03-29
Anticipated expiration: 2041-10-27
Also published as: CN114195884A

Abstract

The present application relates to recombinant human collagen. The invention also provides a method for preparing the recombinant human collagen, and the human collagen which has good water solubility, high expression level and high purity and can obviously improve the wound healing condition is obtained.

Description

Recombinant human collagen and preparation method thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a gene recombinant human collagen and a coding gene thereof, and a preparation method of the recombinant human collagen.

Background

Collagen is a glycoprotein, which is divided into more than 28 kinds of collagen according to different tissue parts, physiological functions and molecular structures, and the most thorough research is conducted on type I, type II and type III collagen. The collagen in human skin is type I collagen and type III collagen, wherein the type I collagen is mainly present in adult skin, tendon and bone tissue, the type III collagen is mainly present in infant skin or vascular intima and intestinal tract, and the type I collagen and the type III collagen are closely related to the skin injury repair process and repair quality. The III type collagen is in a triple helix shape formed by twisting three peptide chains rightward, has excellent biological activity and histocompatibility, can promote fibroblast proliferation of dermis, improves cell activity, is absorbed by fibroblasts as raw materials for synthesizing collagen, stimulates more synthetic collagen of cells, fills and repairs damaged aged skin, reconstructs a reticular structure, enhances the expansion force of the damaged skin, and recovers skin elasticity.

However, natural collagen has a complex structure and is insoluble in water, and these characteristics limit the development, utilization and production of natural collagen. The traditional and most main method for producing the collagen is to treat animal-derived tissues (pigskin, cow hide, donkey hide, fish, etc.) by an acid and alkali method, and extract the collagen from the tissues; in addition, collagen is extracted by an enzyme method; although the methods have higher recovery rate, the collagen prepared by the methods is mixed collagen peptide fragments with different lengths, the peptide fragments have different water solubility, and are difficult to separate into individual peptide fragment pure products, and meanwhile, the collagen is heterologous collagen, so that rejection reaction is expressed clinically, and the application of the collagen as biomedical materials or drug carriers is greatly limited. In addition, the biological activity of the collagen obtained by the method is far less than that of natural collagen of human body, and the collagen cannot meet the requirements of industries such as medicines, foods, cosmetics and the like.

In addition, the collagen obtained by the traditional method is purified in a heterogeneous way, has a certain risk of diseases, and common diseases are swine influenza, mad cow disease, bovine epidemic fever and the like. The FDA in the united states has strict regulatory requirements for disease risk from animal-derived materials or pharmaceuticals, which place higher demands on the development of related products by businesses. The inventor of the invention is based on the defects of the prior art, utilizes the transgenic technology and the gene recombination technology to obtain the recombinant human collagen in animal, plant and microorganism expression systems, solves the defects of hidden danger of viruses and the like existing in the traditional extraction method, and improves the hydrophilicity, immune rejection and the like of the collagen. The present invention is based on this idea.

The COL21A1 gene encodes the alpha chain of type XXI collagen, a member of the family of fasit (fibril-related collagen with interrupted helices) collagens. Type XXI collagen localizes in the tissues containing type I collagen and maintains the integrity of the extracellular matrix. Skin aging is the result of the combined action of endogenous and exogenous factors, but overall, skin aging and the content and structure of collagen in skin are closely related, young skin is smooth and plump, and the main reasons for flexibility and elasticity are just that collagen in dermis is sufficient, and elastic fiber is in the optimal state. The collagen loss and the collagen supplementation can be used for controlling and slowing down the skin aging process and protecting damaged skin, thereby realizing the beauty effect. The inventor of the present invention has unexpectedly obtained a human collagen 21 which has good water solubility, high expression level, high purity and can significantly improve wound healing.

The technical problem solved by the invention is to provide a gene recombinant human collagen and a coding gene thereof; the specific protein amino acid sequence is shown in SEQ ID NO. 1: YVHHHHHHENLYFQGEDGEVRSSCRTAPTDLVFILDGSYSVGPENFEIVKKWLVQITKNFDIGPKFIQVGVVQYSDYPVLEIPLGS YDSGEHLTAAVESILYLGGNTKTGKAIQFALDYLFAKSSRFLTKIAVVLTDGKSQDDVKDAAQAARDSKITLFAIGVGSETEDAE LRAIANKPSSTYVFYVEDYIAISKIREVMKQKLCEESVCPTRIP VAARDERGFDILLGLDVNKKVKKRIQLSPKKIKGYEVTSKVDLSELTSNVFPEGLPPSYVFVSTQRFKVKKIWDLWRILTIDGRPQ IAVTLNGVDKILLFTTTSVINGSQVVTFANPQVKTLFDEGWHQIRLLVTEQDVTLYIDDQQIENKPLHPVLGILINGQTQIGKYSGK EETVQFDVQKLRIYCDPEQNNRETACEIPGFNGECLNGPSDV GSTPAPCICPPGKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLP GFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGT RGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG

In one aspect, the invention relates to recombinant human collagen, which is characterized in that the amino acid sequence is shown as SEQ NO. 1;

in another aspect, the invention relates to a vector for the protein-encoding gene;

in another aspect, the invention relates to a host cell of said protein-encoding gene or said vector;

in another aspect, the invention relates to the use of said recombinant human collagen, said carrier, said host cell for the preparation of a cosmetic, a health product or a medicament;

in another aspect, the present invention relates to a method for producing the recombinant human collagen, which is characterized by comprising the steps of: (1) construction of pichia pastoris genetically engineered bacteria; (2) fermenting and culturing the pichia pastoris genetically engineered bacteria; (3) induction and expression of recombinant human collagen; (4) purification of recombinant human collagen;

in some embodiments, the construction steps of the pichia pastoris genetically engineered bacteria are:

(1) Activated yeast GS115 was streaked on YPD plates;

(2) Selecting monoclonal of activated Pichia pastoris GS115 on the YPD plate, and performing constant-temperature shake culture in YPD liquid;

(3) 100uL of bacterial liquid is sucked by a pipette and inoculated into 100m LYPD liquid, and the bacterial liquid is cultured at the temperature of 30 ℃ and at the speed of 220rpm for 12-13 hours until OD ₆₀₀ ＝1.3-1.5；

(4) Observing whether bacteria are infected or not by microscopic examination, subpackaging thalli by using a sterile 50ml centrifuge tube, and placing the thalli in ice for 10-15min;

(5) Centrifuging for 2 times, discarding supernatant, and re-suspending thallus with sterile water;

(6) Centrifuging for 2 times, discarding supernatant, re-suspending thallus with sorbitol, and packaging to obtain susceptible cell;

(7) Transferring the vector into competent cells by electrotransformation method, and culturing on a plate until cloning;

in some embodiments, the fermentation culture steps of the pichia pastoris genetically engineered bacteria are:

(1) Single colonies on YPD plates were picked up and incubated in a triangular flask containing 50mL BMGY at 30℃and 220rpm for 24h by OD detection ₆₀₀ Confirming the end of the culture;

(2) Centrifuging for 10min, collecting thallus, washing with BMMY once, centrifuging, and diluting thallus concentration to OD ₆₀₀ ＝1.0。

In other embodiments, the recombinant human collagen is induced and expressed as follows:

(1) Transferring the diluted bacterial liquid to a triangular conical flask with the capacity of 500mL, and inducing yeast to express protein at 29 ℃ and 220 rpm; adding methanol every 24 hours to make the final concentration of methanol in the culture medium be 1%, and continuously performing induction culture for 84 hours;

(2) Microscopic examination is carried out to see whether bacteria are infected or not, and the pH value of the fermentation liquor is measured and is required to be less than 6;

(3) Transferring the fermentation broth into a 500mL centrifuge tube with a round bottom, centrifuging at 15000g and 4 ℃ for 20min, and taking a supernatant;

(4) Taking out 1mL of supernatant, adding a corresponding Loading buffer, uniformly mixing, incubating at 95 ℃ for 10min, and preserving at-20 ℃;

in other embodiments, the purification steps of the recombinant human collagen are as follows:

(1) Transferring the obtained fermentation broth supernatant into a conical flask, adding NaCl to make the final concentration of NaCl be 150mM, and adjusting pH of the fermentation broth supernatant to 8.0 by adjusting NaOH or HCl;

(2) 15000g, centrifuging for 30min, ensuring that the supernatant is clear and transparent, and balancing the column with binding buffer of 10 nickel column bed volumes;

(3) Loading samples at a speed of 1mL/min, and collecting the column penetrating fluid while loading the samples;

(4) Eluting unbound proteins and heteroproteins with a binding buffer of 5 nickel column bed volumes;

(5) Eluting the target protein by using an Elutation buffer (500 mM imidazole);

(6) Preparing a protein detection sample: and adding the collected purified proteins, supernatant before column penetration and liquid after column penetration into corresponding Loading buffers respectively, uniformly mixing, incubating at 95 ℃ for 10min, and preserving at-20 ℃.

In other embodiments, the invention relates to recombinant human collagen obtained by the above-described method. The recombinant human collagen obtained by the invention has high purity, can effectively promote cell repair and healing, and has unexpected technical effects compared with the prior art.

Description of the drawings:

fig. 1: recombinant human collagen expression and purification experimental result

Fig. 2: scratch test result of recombinant human collagen

Fig. 3: mouse wound healing experiment

FIG. 3a is a comparative view of wound defect area

FIG. 3b is a line graph of wound diameter change (cure rate) (days 1-7)

FIG. 3c is a bar graph of wound diameter variation (cure rate) (days 1-3)

Detailed Description

Reagent preparation

1. LB broth:

weighing 2.5g LB Broth Powder,100mL ddH ₂ O was dissolved and stored at 121℃for 20min at 4 ℃.

2. BMGY growth medium: 1000mL

3. BMMY induction medium: 1000mL

4. YPD-zeocin plates (500 mL):

450mL ddH ₂ o, 10g of Agarose, 10g of Tryptone, 5g of Yeast Extract, 121 ℃ for 20min. After about 60 ℃,50 mL of 10 XD and zeocin antibiotics were added under a clean bench and stored at 4 ℃.

5、1M Tris-HCl(pH 7.5):

Dissolving 12.1g of Tris base with 80mL of water, cooling the solution, regulating the pH to 7.5 with concentrated HCl, fixing the volume to 100mL with water, sterilizing at 121 ℃ for 20min under high pressure, and preserving at 4 ℃ after cooling.

6、500mM NaCl:

29.22g NaCl was weighed out and dissolved in 1L of water.

7. 500mM imidazole:

34.03g of imidazole was weighed out and dissolved in 1L of water.

8、Binding Buffer:

50mM Tris-HCl 50mL,500mM NaCl 100mL,20mM imidazole 20mL, pH adjusted to 8.0 with HCl or NaOH and then added with water to volume 1000mL.

9、Elution Buffer:

50mM Tris-HCl 50mL,500mM NaCl 100mL,500mM imidazole 500mL, pH adjusted to 8.0 with HCl or NaOH, and then added with water to volume 1000mL.

Definition:

notwithstanding that the numerical ranges and approximations of the parameters set forth in the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range recited as "1 to 10" should be considered to include any and all subranges between (inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood as being incorporated in its entirety.

Example 1: construction of vectors

On the basis of the XXI type collagen tripeptide functional region sequence, 2028bp human XXI type collagen alpha 1 chain gene sequence (col 21A 1) is artificially synthesized, and comprises an enzyme cutting site, a His tag and a signal peptide sequence, and a recombinant plasmid is constructed by taking pPic9k as a carrier. The specific sequence of the vector is shown in SEQ ID NO. 2:

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTT TGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTG CAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACT TTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCA TGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACAC CCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGT CAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATC TCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATAC CGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAAC CCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCC GCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATG ATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTA TCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAAT TGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAGGATCCAAACG ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGA TGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTT CCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTA TTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTACGTAGAATTCCCTAGGGCGG CCGCGAATTAATTCGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTA ATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAG GCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCC TGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTT CCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCGATAAGCTTTAATGCGGTAGTTTAT CACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAA TCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGG GCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTT CTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTG GCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGAT CTATCGAATCTAAATGTAAGTTAAAATCTCTAAATAATTAAATAAGTCCCAGTTTCTCCATACGAACCTTAACAGCATTGCGGT GAGCATCTAGACCTTCAACAGCAGCCAGATCCATCACTGCTTGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCTTGTGA AGTGATGAACTTCTGGAAGGTTGCAGTGTTAACTCCGCTGT ATTGACGGGCATATCCGTACGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTAATCTCCACAACTCTCTGGAGAGTAGGCAC CAACAAACACAGATCCAGCGTGTTGTACTTGATCAACATAAGAAGAAGCATTCTCGATTTGCAGGATCAAGTGTTCAGGAG CGTACTGATTGGACATTTCCAAAGCCTGCTCGTAGGTTGCA ACCGATAGGGTTGTAGAGTGTGCAATACACTTGCGTACAATTTCAACCCTTGGCAACTGCACAGCTTGGTTGTGAACAGCAT CTTCAATTCTGGCAAGCTCCTTGTCTGTCATATCGACAGCCAACAGAATCACCTGGGAATCAATACCATGTTCAGCTTGAGA CAGAAGGTCTGAGGCAACGAAATCTGGATCAGCGTATTTAT CAGCAATAACTAGAACTTCAGAAGGCCCAGCAGGCATGTCAATACTACACAGGGCTGATGTGTCATTTTGAACCATCATCTT GGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAGGAACAGTTTCTGTTCCGTAAGCCATAGCAGCT ACTGCCTGGGCGCCTCCTGCTAGCACGATACACTTAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGGTAAGGGTA CCATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTTGCA ACCAGCAACTTTGGCAGGAACACCCAGCATCAGGGAAGTGGAAGGCAGAATTGCGGTTCCACCAGGAATATAGAGGCCAA CTTTCTCAATAGGTCTTGCAAAACGAGAGCAGACTACACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAGCTT CATGGAATTTCCTGACGTTATCTATAGAGAGATCAATGGCTCTCTTAACGTTATCTGGCAATTGCATAAGTTCCTCTGGGAAA GGAGCTTCTAACACAGGTGTCTTCAAAGCGACTCCATCAA ACTTGGCAGTTAGTTCTAAAAGGGCTTTGTCACCATTTTGACGAACATTGTCGACAATTGGTTTGACTAATTCCATAATCTGT TCCGTTTTCTGGATAGGACGACGAAGGGCATCTTCAATTTCTTGTGAGGAGGCCTTAGAAACGTCAATTTTGCACAATTCAA TACGACCTTCAGAAGGGACTTCTTTAGGTTTGGATTCTTCT TTAGGTTGTTCCTTGGTGTATCCTGGCTTGGCATCTCCTTTCCTTCTAGTGACCTTTAGGGACTTCATATCCAGGTTTCTCTCC ACCTCGTCCAACGTCACACCGTACTTGGCACATCTAACTAATGCAAAATAAAATAAGTCAGCACATTCCCAGGCTATATCTTC CTTGGATTTAGCTTCTGCAAGTTCATCAGCTTCCTCCCTAAT TTTAGCGTTCAACAAAACTTCGTCGTCAAATAACCGTTTGG TATAAGAACCTTCTGGAGCATTGCTCTTACGATCCCACAAGGTGGCTTCCATGGCTCTAAGACCCTTTGATTGGCCAAAACA GGAAGTGCGTTCCAAGTGACAGAAACCAACACCTGTTTGTTCAACCACAAATTTCAAGCAGTCTCCATCACAATCCAATTC GATACCCAGCAACTTTTGAGTTGCTCCAGATGTAGCACCTTTATACCACAAACCGTGACGACGAGATTGGTAGACTCCAGTT TGTGTCCTTATAGCCTCCGGAATAGACTTTTTGGACGAGTA CACCAGGCCCAACGAGTAATTAGAAGAGTCAGCCACCAAAGTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAAGACG CCAACAAAATTTCACTGACAGGGAACTTTTTGACATCTTCA GAAAGTTCGTATTCAGTAGTCAATTGCCGAGCATCAATAATGGGGATTATACCAGAAGCAACAGTGGAAGTCACATCTACC AACTTTGCGGTCTCAGAAAAAGCATAAACAGTTCTACTACC GCCATTAGTGAAACTTTTCAAATCGCCCAGTGGAGAAGAAAAAGGCACAGCGATACTAGCATTAGCGGGCAAGGATGCAA CTTTATCAACCAGGGTCCTATAGATAACCCTAGCGCCTGGGATCATCCTTTGGACAACTCTTTCTGCCAAATCTAGGTCCAA AATCACTTCATTGATACCATTATTGTACAACTTGAGCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACT CAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAAC TTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTGCA ACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCG TATTTTTATTATCAGTGAGTCAGTCATCAGGAGATCCTCTAC GCCGGACGCATCGTGGCCGACCTGCAGGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCA TACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCA GTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGC AAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGT CAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCAT AGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCG TCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAA TCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTAC AAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCA GCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGT AACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCA TCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGA TAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGC GGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGT TCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCTGCAGGT CGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTT CGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCAT GCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAG AGCGTCGAGTATCTATGATTGGAAGTATGGGAATGGTGATA CCCGCATTCTTCAGTGTCTTGAGGTCTCCTATCAGATTATGCCCAACTAAAGCAACCGGAGGAGGAGATTTCATGGTAAATT TCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACTATCTCGGTTATGACAGCAGAAATGTCCTTCTTGGAGACAGT AAATGAAGTCCCACCAATAAAGAAATCCTTGTTATCAGGAA CAAACTTCTTGTTTCGAACTTTTTCGGTGCCTTGAACTATAAAATGTAGAGTGGATATGTCGGGTAGGAATGGAGCGGGCAA ATGCTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTTTGTAGATACTGATGCCAACTTCAGTGACAACGTTGCTATTTCGT TCAAACCATTCCGAATCCAGAGAAATCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACAATAG TGTGCTCGTGTTTTGAGGTCATCTTTGTATGAATAAATCTAG TCTTTGATCTAAATAATCTTGACGAGCCAAGGCGATAAATACCCAAATCTAAAACTCTTTTAAAACGTTAAAAGGACAAGTAT GTCTGCCTGTATTAAACCCCAAATCAGCTCGTAGTCTGATCC TCATCAACTTGAGGGGCACTATCTTGTTTTAGAGAAATTTGCGGAGATGCGATATCGAGAAAAAGGTACGCTGATTTTAAAC GTGAAATTTATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGA TGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAG CCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATA CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG TGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGG AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTC GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAA GAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGT ATCACGAGGCCCTTTCGTCTTCAAGAATTAATTCTCATGTTTGACAGCTTATCATCGATAAGCTGACTCATGTTGGTATTGTGA AATAGACGCAGATCGGGAACACTGAAAAATAACAGTTATTA TTCG

example 2: expression and purification procedure of recombinant collagen

2.1 expression of recombinant collagen

1. Plasmid extraction

Colonies were picked from the puncture bacteria delivered by the company with a gun head, streaked on Amp+ plates, and incubated in an incubator at 37℃for 12h. Single colony is selected and cultured in 400 mu L of Amp+LB culture solution at 37 ℃ and 280rpm for 7 hours, then transferred to 100mL of Amp+LB culture solution for expansion culture, and operated according to the instruction of a medium extraction kit (Tiangen) after 18 hours, and finally the concentration and OD value are measured by using a Nanodrop 2000 and stored at-20 ℃.

2. Plasmid linearization enzyme digestion

200. Mu.L of SacI cleavage system (buffer 20. Mu.L of enzyme 5. Mu.L of plasmid 20. Mu.g) was prepared and digested overnight, and the fragments were purified by phenol chloroform extraction.

3. Yeast competent preparation

(1) Activated yeast GS115 was cultured at 30℃for 48-72 hours on YPD plates.

(2) The activated Pichia pastoris GS115 monoclonal on YPD plates was selected and cultured in 5ml of YPD liquid at a constant temperature of 30℃and 220rpm for 24h.

(3) 100uL of bacterial liquid is sucked by a liquid transferring gun and inoculated into 100mLYPD liquid, the temperature is 30 ℃, the rpm is 220, the time is 12-13 hours, and the culture time is up to 12 hours, and the open measurement 0D600,0D600 is strictly controlled to be 1.3-1.5;

(4) Observing the presence or absence of bacteria by microscopic examination, subpackaging the thalli with 4 sterile 50ml centrifuge tubes, and placing the thalli on ice for 10-15min;

(5) 1500 Xg, 4 ℃, centrifuge for 5min, discard supernatant, resuspend cells with 25mL ice-cold sterile water for each centrifuge tube;

(6) Centrifuging at 4deg.C for 5min at 1500 Xg, and discarding supernatant; 15mL (2/3) of ice-cold sterile water per tube was used to resuspend the cells;

(7) 1500 Xg, 4 ℃, centrifuge for 5min, discard supernatant, 5ml ice-cold 1M sorbitol re-suspension cells per tube;

(8) 1500 Xg, 4 ℃, centrifuge for 5min, discard supernatant, add 600uL ice cold sorbitol heavy suspension, final volume about 1mL. The 1mL sorbitol is split charging to obtain competent cells, which can be directly used for electrotransformation.

4. Yeast electric conversion

(1) The electric excitation cup is dried and then placed in ice, competent cells and plasmids are placed in ice for melting, and 1mL of sorbitol is taken for precooling.

(2) 100. Mu.L of competent plasmid was mixed with 3-20. Mu.g of recovered linearized plasmid (volume less than 20. Mu.L) and transferred to a pre-chilled 0.2cm electrorotating cup.

(3) Electrical conversion 2000v, capacitance 25VF, resistance 200 ohm.

(4) Immediately adding 600-700 μl of pre-cooled sorbitol into the cup, mixing well, and standing on ice for 5min.

(5) The contents of the electrorotor were transferred to an aseptic 1.5mL EP tube in an ultra clean bench and left to stand in an incubator at 30℃for 1h

(6) 2 parts of the mixture was spread on a plate (plate was preheated in an incubator at 30 ℃).

(7) Culturing at 30deg.C until clone is produced

5. Pichia pastoris culture and exogenous protein induction expression

(A) The steps are as follows:

(1) Single colonies on YPD plates were picked with a 10. Mu.L tip in a Erlenmeyer flask containing 50mL BMGY, at 30℃and 220rpm for 24h.

(2) Performing microscopic examination; OD measurement ₆₀₀ Values.

(3) 3000g, centrifuging at 24℃for 10min, collecting the cells, washing once with BMMY, and centrifuging again. According to the previous stepThe OD and volume values were measured, and the cell concentration was diluted to 0D with BMMY ₆₀₀ ＝1.0。

(4) The diluted bacterial liquid was transferred to a 500mL Erlenmeyer flask at 29℃and 220rpm to induce yeast expression proteins. Then adding methanol into the ultra-clean workbench every 24 hours to make the final concentration of the methanol in the culture medium be 1%.

(5) The culture was continuously induced for 84 hours.

(6) And (3) microscopic examination is carried out to see whether bacteria are infected or not, and the pH value of the fermentation liquor is measured and is required to be less than 6.

(7) The fermentation broth was transferred to a 500mL round bottom centrifuge tube, 15000g, centrifuged at 4℃for 20min and the supernatant was taken.

(8) Taking out 1mL of supernatant, adding a corresponding Loading buffer, uniformly mixing, incubating at 95 ℃ for 10min, and preserving at-20 ℃.

Results display (fig. 1): the expression result (lane 3) meets the requirements of further experiments, and the culture yield is 16mg/L.

2.2 purification

And A, step A:

(1) The resulting broth supernatant was transferred to a conical flask and NaCl was added to give a final concentration of 150mM NaCl.

(2) Adjusting pH of the supernatant of the fermentation broth to 8.0 by adjusting NaOH or HCl.

(3) 15000g, centrifugation for 30min, ensures clear and transparent supernatant, which would otherwise block the AKTA sampling tube.

(4) The column was equilibrated with a binding buffer of 10 nickel column bed volumes.

(5) Loading at a speed of 1mL/min, and collecting the column penetrating liquid while loading so as to prevent protein loss which is not bonded to the nickel column.

(6) Unbound proteins and heteroproteins were eluted with a binding buffer of 5 nickel column bed volumes.

(7) The target protein was eluted with an Elutation buffer (500 mM imidazole), and a tube was collected every 5mL, approximately 20mL, depending on AU values.

(8) Preparing a protein detection sample: and adding the collected purified proteins, supernatant before column penetration and liquid after column penetration into corresponding Loading buffers respectively, uniformly mixing, incubating at 95 ℃ for 10min, and preserving at-20 ℃.

As shown in FIG. 1 (lane 3), the purification effect was good, the bands were relatively uniform, and the concentration of recombinant XXI-type collagen was determined to be 0.28. Mu. Mol/L and 0.56. Mu. Mol/L, which meet the basic requirements of further experiments.

EXAMPLE 3 recombinant collagen scoring procedure and conclusion

3.1 step

(1) And (3) paving: cells in logarithmic growth phase, trypsinized into single cell suspension, inoculated in 6 well plates, 3 wells repeated per group; cell plating 6×105 cells/well, ensuring that the cells will grow over the next day, 2 mL total medium per well;

(2) Cell culture at 37℃with 5% CO ₂ Culturing in an incubator for 24 hours;

(3) Scoring: the gun head is vertical compared with a ruler in the next day, and the same gun head is used between different holes;

(4) Cleaning: washing cells 3 times with PBS, and adding a culture medium containing 2% of serum;

(5) Taking fixed upper, lower, left and right four points of a cross shape from each hole, photographing under a 4-time mirror, and taking samples according to time points of 0h and 48h, wherein the background is consistent;

(6) Image data analysis

As can be seen from FIG. 2, after 48h of culture, recombinant XXI-type collagen at different concentrations can significantly promote migration of HaCaT cells, in contrast to the effect of COL21 at 0.28. Mu. Mol/L, which is similar to the effect of 0.56. Mu. Mol/L.

Example 4: recombinant collagen mouse skin injury repair experiment

4.1 construction of full-layer skin incision model of mice

24C 57BL/6 mice with the age of 7W are taken to establish a skin full-layer injury model, 5% Liu Mianning is used for carrying out intraperitoneal injection anesthesia at the dosage of 100 mu L, the back hair of the mice is removed by paraffin, an annular ring is printed at the middle position of the back (preventing the mice from licking to wounds) by an 8mm leather drill, the central skin of the annular ring is clamped by forceps, the full-layer skin is sheared by tissue scissors along the annular ring, a mechanical injury wound surface is formed, and the model is prepared when the astronomical is day 0. After the molding is completed, single-cage feeding is carried out.

4.2 therapeutic intervention in mice

The 24 model mice were randomly divided into 3 groups of 8 mice. The treatment is started on the same day when the mould is made, 120 mu L of liquid medicine is respectively sucked by a liquid transfer device, the liquid medicine is slowly dripped on the wound surface, the mice are put back into a feeding cage after the liquid medicine is completely absorbed, the treatment is carried out for 2 times a day, and the treatment is carried out for 11 days continuously.

4.3 wound diameter healing Rate statistics

Wound diameter was measured with vernier calipers on days 1, 3, 5 and 7 of molding, and three measurements were repeated for each mouse. Healing rate = (first day wound diameter size-post-treatment diameter size)/first day wound diameter size x 100%.

4.4 photographing to observe the wound healing condition

Cameras were used to record wound recovery from each group of mice on days 1, 3, 5, and 7 of molding, respectively.

The experimental group was dosed at 0.4mg/mL (COL 21) and 240 μl of PBS (control) or therapeutic liquid was added daily to the wound on the day of modeling for 11 consecutive days. As can be seen from fig. 3 (fig. a), the wound defect areas of the control group and the experimental group were continuously reduced with the increase of the treatment days, but the wound area of the COL21 group was significantly reduced compared with the control group. When the wound diameter change was quantitatively analyzed, it was found (fig. b, c) that the healing rate was significantly higher in the first 3 days of COL21 group than in the control group (P < 0.001), and there was no significant difference in other time periods, presumably the drug concentration was too low, and as the wound was continuously epithelialized, the external drug was difficult to enter deep into the cortex, and thus higher concentration was required to function.

The above description is of preferred embodiments only, which are exemplary only and not limiting of the combination of features necessary to practice the invention. The headings provided are not meant to limit the various embodiments of the invention. Terms such as "comprising," "including," and "comprising" are not intended to be limiting. Furthermore, unless otherwise indicated, the absence of a numerical modification includes the plural form, and "or", "or" means "and/or". Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Sequence listing

<110> Hemei biotechnology (Zhejiang) Co., ltd

<120> a recombinant human collagen and method for preparing the same

<130> 1

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 673

<212> PRT

<213> Artificial sequence (unknown)

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Tyr Val His His His His His His Glu Asn Leu Tyr Phe Gln Gly Glu

1 5 10 15

Asp Gly Glu Val Arg Ser Ser Cys Arg Thr Ala Pro Thr Asp Leu Val

20 25 30

Phe Ile Leu Asp Gly Ser Tyr Ser Val Gly Pro Glu Asn Phe Glu Ile

35 40 45

Val Lys Lys Trp Leu Val Gln Ile Thr Lys Asn Phe Asp Ile Gly Pro

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Lys Phe Ile Gln Val Gly Val Val Gln Tyr Ser Asp Tyr Pro Val Leu

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Glu Ile Pro Leu Gly Ser Tyr Asp Ser Gly Glu His Leu Thr Ala Ala

85 90 95

Val Glu Ser Ile Leu Tyr Leu Gly Gly Asn Thr Lys Thr Gly Lys Ala

100 105 110

Ile Gln Phe Ala Leu Asp Tyr Leu Phe Ala Lys Ser Ser Arg Phe Leu

115 120 125

Thr Lys Ile Ala Val Val Leu Thr Asp Gly Lys Ser Gln Asp Asp Val

130 135 140

Lys Asp Ala Ala Gln Ala Ala Arg Asp Ser Lys Ile Thr Leu Phe Ala

145 150 155 160

Ile Gly Val Gly Ser Glu Thr Glu Asp Ala Glu Leu Arg Ala Ile Ala

165 170 175

Asn Lys Pro Ser Ser Thr Tyr Val Phe Tyr Val Glu Asp Tyr Ile Ala

180 185 190

Ile Ser Lys Ile Arg Glu Val Met Lys Gln Lys Leu Cys Glu Glu Ser

195 200 205

Val Cys Pro Thr Arg Ile Pro Val Ala Ala Arg Asp Glu Arg Gly Phe

210 215 220

Asp Ile Leu Leu Gly Leu Asp Val Asn Lys Lys Val Lys Lys Arg Ile

225 230 235 240

Gln Leu Ser Pro Lys Lys Ile Lys Gly Tyr Glu Val Thr Ser Lys Val

245 250 255

Asp Leu Ser Glu Leu Thr Ser Asn Val Phe Pro Glu Gly Leu Pro Pro

260 265 270

Ser Tyr Val Phe Val Ser Thr Gln Arg Phe Lys Val Lys Lys Ile Trp

275 280 285

Asp Leu Trp Arg Ile Leu Thr Ile Asp Gly Arg Pro Gln Ile Ala Val

290 295 300

Thr Leu Asn Gly Val Asp Lys Ile Leu Leu Phe Thr Thr Thr Ser Val

305 310 315 320

Ile Asn Gly Ser Gln Val Val Thr Phe Ala Asn Pro Gln Val Lys Thr

325 330 335

Leu Phe Asp Glu Gly Trp His Gln Ile Arg Leu Leu Val Thr Glu Gln

340 345 350

Asp Val Thr Leu Tyr Ile Asp Asp Gln Gln Ile Glu Asn Lys Pro Leu

355 360 365

His Pro Val Leu Gly Ile Leu Ile Asn Gly Gln Thr Gln Ile Gly Lys

370 375 380

Tyr Ser Gly Lys Glu Glu Thr Val Gln Phe Asp Val Gln Lys Leu Arg

385 390 395 400

Ile Tyr Cys Asp Pro Glu Gln Asn Asn Arg Glu Thr Ala Cys Glu Ile

405 410 415

Pro Gly Phe Asn Gly Glu Cys Leu Asn Gly Pro Ser Asp Val Gly Ser

420 425 430

Thr Pro Ala Pro Cys Ile Cys Pro Pro Gly Lys Pro Gly Leu Gln Gly

435 440 445

Pro Lys Gly Asp Pro Gly Leu Pro Gly Asn Pro Gly Tyr Pro Gly Gln

450 455 460

Pro Gly Gln Asp Gly Lys Pro Gly Tyr Gln Gly Ile Ala Gly Thr Pro

465 470 475 480

Gly Val Pro Gly Ser Pro Gly Ile Gln Gly Ala Arg Gly Leu Pro Gly

485 490 495

Tyr Lys Gly Glu Pro Gly Arg Asp Gly Asp Lys Gly Asp Arg Gly Leu

500 505 510

Pro Gly Phe Pro Gly Leu His Gly Met Pro Gly Ser Lys Gly Glu Met

515 520 525

Gly Ala Lys Gly Asp Lys Gly Ser Pro Gly Phe Tyr Gly Lys Lys Gly

530 535 540

Ala Lys Gly Glu Lys Gly Asn Ala Gly Phe Pro Gly Leu Pro Gly Pro

545 550 555 560

Ala Gly Glu Pro Gly Arg His Gly Lys Asp Gly Leu Met Gly Ser Pro

565 570 575

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

580 585 590

Thr Arg Gly Glu Pro Gly Ile Pro Gly Phe Pro Gly Asn Arg Gly Leu

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Met Gly Gln Lys Gly Glu Ile Gly Pro Pro Gly Gln Gln Gly Lys Lys

610 615 620

Gly Ala Pro Gly Met Pro Gly Leu Met Gly Ser Asn Gly Ser Pro Gly

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Gln Pro Gly Thr Pro Gly Ser Lys Gly Ser Lys Gly Glu Pro Gly Ile

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Gln Gly Met Pro Gly Ala Ser Gly Leu Lys Gly Glu Pro Gly Ala Thr

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Gly

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<213> Artificial sequence (unknown)

<400> 2

agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300

tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360

agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420

gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480

ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt 540

cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600

ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660

ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720

gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780

atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840

actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900

caacttgaga agatcaaaaa acaactaatt attcgaagga tccaaacgat gagatttcct 960

tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact 1020

acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta 1080

gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg 1140

tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa 1200

agagaggctg aagcttacgt agaattccct agggcggccg cgaattaatt cgccttagac 1260

atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa 1320

tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt 1380

ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc 1440

ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa 1500

tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta 1560

agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt 1620

taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc 1680

tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc 1740

tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc 1800

tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg 1860

gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg 1920

cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat 1980

taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct 2040

tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg 2100

tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca 2160

tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga 2220

gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca 2280

ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc 2340

tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc 2400

cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct 2460

gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga 2520

aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa 2580

ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg 2640

gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct 2700

gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca 2760

ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga 2820

gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa 2880

gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt 2940

gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga 3000

gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct 3060

ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact 3120

ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg 3180

acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca 3240

tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct 3300

tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc 3360

ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc 3420

tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca 3480

cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta 3540

attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga 3600

gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca 3660

aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc 3720

aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta 3780

gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata 3840

gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca 3900

gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt 3960

tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga 4020

gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg 4080

gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc 4140

agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca 4200

accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc 4260

aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg 4320

atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc 4380

tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg 4440

gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc 4500

aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg 4560

tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc 4620

gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca 4680

taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga 4740

gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct 4800

gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa 4860

caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca 4920

attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat 4980

tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc 5040

agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 5100

tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 5160

tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa 5220

caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 5280

gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 5340

gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat 5400

caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc 5460

atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 5520

gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 5580

tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 5640

gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 5700

atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac 5760

tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt 5820

aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca 5880

ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc 5940

gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg 6000

tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc 6060

tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc 6120

gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga 6180

ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt 6240

tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag 6300

aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag 6360

gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata 6420

tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg 6480

gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg 6540

aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac 6600

tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc 6660

taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt 6720

aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat 6780

caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa 6840

ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga 6900

tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 6960

ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 7020

cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca 7080

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 7140

aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 7200

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 7260

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 7320

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 7380

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 7440

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 7500

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 7560

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 7620

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 7680

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 7740

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 7800

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 7860

aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 7920

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 7980

gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 8040

cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 8100

gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 8160

tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 8220

ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 8280

ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 8340

atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 8400

cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct 8460

tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 8520

aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 8580

tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 8640

ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 8700

agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa 8760

gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 8820

agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 8880

accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 8940

gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 9000

cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 9060

ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 9120

atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt 9180

ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc 9240

agatcgggaa cactgaaaaa taacagttat tattcg 9276

Claims

1. A recombinant human collagen is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A vector comprising the protein-encoding gene of claim 1.

3. A host cell comprising the protein-encoding gene of claim 1 or the vector of claim 2.

4. Use of the recombinant human collagen according to claim 1, the vector according to claim 2, the host cell according to claim 3 for the preparation of a cosmetic or pharmaceutical for improving wound healing.