CN114195884A - Recombinant human collagen and preparation method thereof - Google Patents

Abstract

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

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 classified into more than 28 types of collagen according to differences in tissue sites, physiological functions and molecular structures, and the types of collagen, I, II and III, which are the most thoroughly studied, account for the most. 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 tissues, the type III collagen is mainly present in infant skin or blood vessel 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 has three strands of helices twisted by three peptide chains rightward, has excellent biological activity and histocompatibility, can promote fibroblast proliferation of a dermis layer, improve cell activity, is absorbed by fibroblast serving as a raw material for synthesizing the collagen, stimulates cells to synthesize more collagen, fills and repairs damaged and aged skin, reconstructs a reticular structure, enhances the expansion force of the damaged skin, and restores the elasticity of the skin.

However, the structure of native collagen is complex and insoluble in water, and these characteristics limit the development, utilization and production of native collagen. The traditional and most important method for producing collagen is to treat tissues of animal origin (pigskin, cow skin, donkey skin, fish, etc.) by acid and alkali methods, and to extract collagen therefrom; in addition, the method also comprises the steps of extracting collagen by an enzyme method; although the methods have higher recovery rate, the collagens prepared by the methods are all mixed collagen peptide fragments with different lengths, the water solubility of each peptide fragment is not uniform, and the pure products of the single peptide fragments are difficult to separate. In addition, the biological activity of the collagen obtained by the method is far lower than that of natural collagen of a human body, and the industrial requirements of medicines, foods, cosmetics and the like cannot be met.

In addition, the collagen obtained by the traditional method is heterogeneously purified, has certain disease risk, and common diseases comprise swine influenza, mad cow disease, bovine epidemic fever and the like. The FDA in the united states has strict regulatory requirements on the disease risk brought by animal-derived materials or drugs, which puts higher demands on the development of related products for enterprises. The inventor of the invention obtains the recombinant human collagen in animal, plant and microorganism expression systems by utilizing a transgenic technology and a gene recombination technology based on the defects of the prior art, overcomes the defects of virus hidden danger and the like existing in the traditional extraction method, and simultaneously improves the hydrophilicity, the immunological rejection and the like of the collagen. The present invention has been made based on this idea.

The COL21a1 gene encodes the α chain of collagen type XXI, which is a member of the FACIT (fibril associated collagen with interrupted helices) collagen family. Type XXI collagen localizes in type I collagen-containing tissues 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 is closely related to the content and structure of collagen in skin, young skin is smooth and plump, and the main reason for flexibility and elasticity is that the collagen in the dermis is sufficient, and elastic fibers are in the optimal state. Slowing down collagen loss and supplementing collagen can be used for controlling and slowing down the skin aging process and protecting damaged skin, thereby realizing the beautifying effect. The inventor of the invention unexpectedly obtains the human collagen 21 which has good water solubility, high expression level and high purity and can obviously improve the wound healing condition.

The technical problem to be 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 as SEQ ID NO. 1: YVHHHHHHENLYFQGEDGEVRSSCRTAPTDLVFILDGSYSVG PENFEIVKKWLVQITKNFDIGPKFIQVGVVQYSDYPVLEIPLGS YDSGEHLTAAVESILYLGGNTKTGKAIQFALDYLFAKSSRFLT KIAVVLTDGKSQDDVKDAAQAARDSKITLFAIGVGSETEDAE LRAIANKPSSTYVFYVEDYIAISKIREVMKQKLCEESVCPTRIP VAARDERGFDILLGLDVNKKVKKRIQLSPKKIKGYEVTSKVD LSELTSNVFPEGLPPSYVFVSTQRFKVKKIWDLWRILTIDGRPQ IAVTLNGVDKILLFTTTSVINGSQVVTFANPQVKTLFDEGWHQ IRLLVTEQDVTLYIDDQQIENKPLHPVLGILINGQTQIGKYSGK EETVQFDVQKLRIYCDPEQNNRETACEIPGFNGECLNGPSDV GSTPAPCICPPGKPGLQGPKGDPGLPGNPGYPGQPGQDGKPG YQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLP GFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNA GFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGT RGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMG SNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG

In one aspect, the invention relates to a 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 of a gene encoding said protein;

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

in another aspect, the invention relates to the use of the recombinant human collagen, the vector, and the host cell in the preparation of cosmetics, health products, or drugs;

in another aspect, the present invention relates to a method for producing recombinant human collagen, which comprises the following steps: (1) constructing pichia pastoris gene engineering bacteria; (2) fermentation culture of pichia pastoris gene engineering bacteria; (3) inducing and expressing the recombinant human collagen; (4) purifying the recombinant human collagen;

in some embodiments, the pichia genetic engineering bacteria are constructed by the following steps:

(1) streak-culturing activated yeast GS115 on YPD plates;

(2) selecting a single clone of pichia pastoris GS115 activated on the YPD flat plate to be placed in the YPD liquid, and carrying out constant-temperature shaking culture;

(3) the liquid transfer gun sucks 100uL of bacterial liquid, inoculates the bacterial liquid in 100m LYPD liquid, the temperature is 30 ℃, the rpm is 220, 12-13h, and the culture is carried out until OD is reached₆₀₀＝1.3-1.5；

(4) Observing whether there is contamination by microscopic examination, subpackaging thallus with sterile 50ml centrifuge tube, and standing on ice for 10-15 min;

(5) centrifuging for 2 times, discarding supernatant, and resuspending thallus with sterile water;

(6) centrifuging for 2 times, discarding supernatant, resuspending thallus with sorbitol, and packaging to obtain competent cells;

(7) transferring the carrier into competent cells by an electric transfer method, and then performing plate culture until cloning is generated;

in some embodiments, the fermentation culture step of the pichia pastoris genetically engineered bacteria is:

(1) single colonies on YPD plates were picked and grown at 30 ℃ and 220rpm for 24h in a conical flask containing 50mL of BMGY, by measuring OD₆₀₀Confirming the end of the culture;

(2) centrifuging for 10min, collecting thallus, washing with BMMY, centrifuging, and diluting thallus concentration to OD₆₀₀＝1.0。

In other embodiments, the steps of inducing and expressing the recombinant human collagen are as follows:

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

(2) microscopic examination, namely, observing whether the bacteria are infected or not, and measuring the pH value of the fermentation liquor, wherein the pH value is less than 6;

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

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

in other embodiments, the recombinant human collagen is purified by the following steps:

(1) transferring the obtained supernatant of the fermentation liquor into a conical flask, adding NaCl to ensure that the final concentration of the NaCl is 150mM, and adjusting the pH of the supernatant of the fermentation liquor to 8.0 by adjusting NaOH or HCl;

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

(3) sampling at the speed of 1mL/min, and collecting the column-penetrating liquid while sampling;

(4) eluting unbound proteins and hetero-proteins with binding buffers of 5 nickel column bed volumes;

(5) eluting the target protein by using Elution buffer (500mM imidazole);

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

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

Description of the drawings:

FIG. 1: experimental result of expression and purification of recombinant human collagen

FIG. 2: scratch experimental result of recombinant human collagen

FIG. 3: wound healing experiments in mice

FIG. 3a is a comparison of wound defect areas

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

FIG. 3c histogram of wound diameter change (cure rate) (days 1-3)

Detailed Description

Reagent preparation

1. LB broth culture medium:

weighing 2.5g LB Broth Powder, 100mL ddH₂Dissolving O, and storing at 121 deg.C for 20min and 4 deg.C.

2. BMGY growth medium: 1000mL

3. BMMY induction medium: 1000mL

4. YPD-zeocin plates (500 mL):

450mL ddH₂o, 10g Agarose, 10g Tryptone, 5g Yeast Extract, 121 ℃, 20 min. After 60 ℃ approximately 50mL of 10 XD and zeocin antibiotic were added under a clean bench, and stored at 4 ℃.

5、1M Tris-HCl(pH 7.5):

Dissolving 12.1g Tris alkali in 80mL water, adjusting pH to 7.5 with concentrated HCl after the solution is cooled, adding water to constant volume of 100mL, autoclaving at 121 deg.C for 20min, cooling, and storing at 4 deg.C.

6、500mM NaCl:

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

7. 500mM imidazole:

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

8、Binding Buffer:

50mL of 50mM Tris-HCl, 100mL of 500mM NaCl and 20mL of 20mM imidazole, adjusting the pH to 8.0 by using HCl or NaOH, and adding water to the solution to reach the volume of 1000 mL.

9、Elution Buffer:

50mL of 50mM Tris-HCl, 100mL of 500mM NaCl and 500mL of 500mM imidazole, adjusting the pH to 8.0 by using HCl or NaOH, and adding water to the solution to reach 1000 mL.

Defining:

notwithstanding that the numerical ranges and parameter approximations set forth 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 measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and 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. Additionally, any reference that is said to be "incorporated herein" is to 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, a 2028bp human XXI type collagen alpha 1 chain gene sequence (col21A1) is artificially synthesized, the sequence comprises an enzyme cutting site, a His label and a signal peptide sequence, and a recombinant plasmid is constructed by taking pPic9k as a vector. The specific sequence of the vector is shown as SEQ ID NO. 2:

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTT TGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGC CAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTG CAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACT TTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGG AGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCA TGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTT CATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACAC CCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGT CAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGT TTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATC TCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAAC GGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATAC CGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATA ATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAAC CCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCC GCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCC AAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATG ATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATA TAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTA TCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAAT TGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGA GAAGATCAAAAAACAACTAATTATTCGAAGGATCCAAACG ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCA TCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGA TGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACT CAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTT CCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTA TTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAG AAAAGAGAGGCTGAAGCTTACGTAGAATTCCCTAGGGCGG CCGCGAATTAATTCGCCTTAGACATGACTGTTCCTCAGTTC AAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTA ATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAG GCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAG GATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCC TGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGG GTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTT CCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTC GTTTGTGCAAGCTTATCGATAAGCTTTAATGCGGTAGTTTAT CACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAA TCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCT GGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGG GCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGT CACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTT CTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTG GCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACT ATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGAT CTATCGAATCTAAATGTAAGTTAAAATCTCTAAATAATTAAAT AAGTCCCAGTTTCTCCATACGAACCTTAACAGCATTGCGGT GAGCATCTAGACCTTCAACAGCAGCCAGATCCATCACTGCT TGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCTTGTGA AGTGATGAACTTCTGGAAGGTTGCAGTGTTAACTCCGCTGT ATTGACGGGCATATCCGTACGTTGGCAAAGTGTGGTTGGTA CCGGAGGAGTAATCTCCACAACTCTCTGGAGAGTAGGCAC CAACAAACACAGATCCAGCGTGTTGTACTTGATCAACATAA GAAGAAGCATTCTCGATTTGCAGGATCAAGTGTTCAGGAG CGTACTGATTGGACATTTCCAAAGCCTGCTCGTAGGTTGCA ACCGATAGGGTTGTAGAGTGTGCAATACACTTGCGTACAAT TTCAACCCTTGGCAACTGCACAGCTTGGTTGTGAACAGCAT CTTCAATTCTGGCAAGCTCCTTGTCTGTCATATCGACAGCC AACAGAATCACCTGGGAATCAATACCATGTTCAGCTTGAGA CAGAAGGTCTGAGGCAACGAAATCTGGATCAGCGTATTTAT CAGCAATAACTAGAACTTCAGAAGGCCCAGCAGGCATGTC AATACTACACAGGGCTGATGTGTCATTTTGAACCATCATCTT GGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGT CACACTTAGGAACAGTTTCTGTTCCGTAAGCCATAGCAGCT ACTGCCTGGGCGCCTCCTGCTAGCACGATACACTTAGCACC AACCTTGTGGGCAACGTAGATGACTTCTGGGGTAAGGGTA CCATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTTGCA ACCAGCAACTTTGGCAGGAACACCCAGCATCAGGGAAGTG GAAGGCAGAATTGCGGTTCCACCAGGAATATAGAGGCCAA CTTTCTCAATAGGTCTTGCAAAACGAGAGCAGACTACACC AGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAGCTT CATGGAATTTCCTGACGTTATCTATAGAGAGATCAATGGCTC TCTTAACGTTATCTGGCAATTGCATAAGTTCCTCTGGGAAA GGAGCTTCTAACACAGGTGTCTTCAAAGCGACTCCATCAA ACTTGGCAGTTAGTTCTAAAAGGGCTTTGTCACCATTTTGA CGAACATTGTCGACAATTGGTTTGACTAATTCCATAATCTGT TCCGTTTTCTGGATAGGACGACGAAGGGCATCTTCAATTTC TTGTGAGGAGGCCTTAGAAACGTCAATTTTGCACAATTCAA TACGACCTTCAGAAGGGACTTCTTTAGGTTTGGATTCTTCT TTAGGTTGTTCCTTGGTGTATCCTGGCTTGGCATCTCCTTTC CTTCTAGTGACCTTTAGGGACTTCATATCCAGGTTTCTCTCC ACCTCGTCCAACGTCACACCGTACTTGGCACATCTAACTAA TGCAAAATAAAATAAGTCAGCACATTCCCAGGCTATATCTTC CTTGGATTTAGCTTCTGCAAGTTCATCAGCTTCCTCCCTAAT TTTAGCGTTCAACAAAACTTCGTCGTCAAATAACCGTTTGG TATAAGAACCTTCTGGAGCATTGCTCTTACGATCCCACAAG GTGGCTTCCATGGCTCTAAGACCCTTTGATTGGCCAAAACA GGAAGTGCGTTCCAAGTGACAGAAACCAACACCTGTTTGT TCAACCACAAATTTCAAGCAGTCTCCATCACAATCCAATTC GATACCCAGCAACTTTTGAGTTGCTCCAGATGTAGCACCTT TATACCACAAACCGTGACGACGAGATTGGTAGACTCCAGTT TGTGTCCTTATAGCCTCCGGAATAGACTTTTTGGACGAGTA CACCAGGCCCAACGAGTAATTAGAAGAGTCAGCCACCAAA GTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAAGACG CCAACAAAATTTCACTGACAGGGAACTTTTTGACATCTTCA GAAAGTTCGTATTCAGTAGTCAATTGCCGAGCATCAATAAT GGGGATTATACCAGAAGCAACAGTGGAAGTCACATCTACC AACTTTGCGGTCTCAGAAAAAGCATAAACAGTTCTACTACC GCCATTAGTGAAACTTTTCAAATCGCCCAGTGGAGAAGAA AAAGGCACAGCGATACTAGCATTAGCGGGCAAGGATGCAA CTTTATCAACCAGGGTCCTATAGATAACCCTAGCGCCTGGG ATCATCCTTTGGACAACTCTTTCTGCCAAATCTAGGTCCAA AATCACTTCATTGATACCATTATTGTACAACTTGAGCAAGTT GTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACT CAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAAC TTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACA CGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTGCA ACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGA AGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCG TATTTTTATTATCAGTGAGTCAGTCATCAGGAGATCCTCTAC GCCGGACGCATCGTGGCCGACCTGCAGGGGGGGGGGGGG CGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCA TACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGA GGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCA GTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCT GCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGC AAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGT CAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATT CTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCAT AGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCG TCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAAT AAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAA TCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGAC TTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGA GCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTAC AAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCA GCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTA ATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGT AACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGT CGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCA TCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTT TCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGA TAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCAT TTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGC GGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAAC ACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGT TCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGA TTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCTGCAGGT CGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATA TCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTT CGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAG GCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCAT GCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCT ACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAG AGCGTCGAGTATCTATGATTGGAAGTATGGGAATGGTGATA CCCGCATTCTTCAGTGTCTTGAGGTCTCCTATCAGATTATGC CCAACTAAAGCAACCGGAGGAGGAGATTTCATGGTAAATT TCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACT ATCTCGGTTATGACAGCAGAAATGTCCTTCTTGGAGACAGT AAATGAAGTCCCACCAATAAAGAAATCCTTGTTATCAGGAA CAAACTTCTTGTTTCGAACTTTTTCGGTGCCTTGAACTATAA AATGTAGAGTGGATATGTCGGGTAGGAATGGAGCGGGCAA ATGCTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTTTGT AGATACTGATGCCAACTTCAGTGACAACGTTGCTATTTCGT TCAAACCATTCCGAATCCAGAGAAATCAAAGTTGTTTGTCT ACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACAATAG TGTGCTCGTGTTTTGAGGTCATCTTTGTATGAATAAATCTAG TCTTTGATCTAAATAATCTTGACGAGCCAAGGCGATAAATAC CCAAATCTAAAACTCTTTTAAAACGTTAAAAGGACAAGTAT GTCTGCCTGTATTAAACCCCAAATCAGCTCGTAGTCTGATCC TCATCAACTTGAGGGGCACTATCTTGTTTTAGAGAAATTTG CGGAGATGCGATATCGAGAAAAAGGTACGCTGATTTTAAAC GTGAAATTTATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGA TGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAG CCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG CGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATA CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG TGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGG AGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT CAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGG AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAC GTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTC GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC GACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGC AAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAA GAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGT ATCACGAGGCCCTTTCGTCTTCAAGAATTAATTCTCATGTTT GACAGCTTATCATCGATAAGCTGACTCATGTTGGTATTGTGA AATAGACGCAGATCGGGAACACTGAAAAATAACAGTTATTA TTCG

example 2: recombinant collagen expression and purification procedure

2.1 expression of recombinant collagen

1. Plasmid extraction

Colonies were picked from among the puncturing bacteria delivered from the company using a pipette tip, streaked on an Amp + plate, and cultured in a 37 ℃ incubator for 12 hours. Picking single colony in 400 μ L Amp + LB culture solution, shaking culturing at 37 deg.C and 280rpm for 7h, transferring to 100mL Amp + LB culture solution for amplification culture, operating according to the instruction of the extraction kit (Tiangen) after 18h, and finally determining concentration and OD value by using Nanodrop 2000 and storing at-20 deg.C.

2. Plasmid linearized digestion

200. mu.L of SacI digestion system (buffer 20. mu.L of enzyme 5. mu.L of plasmid 20. mu.g) was prepared and digested overnight, and the fragment was purified by phenol chloroform extraction.

3. Yeast competent preparation

(1) The activated yeast GS115 was streaked on YPD plates and cultured at 30 ℃ for 48-72 hours.

(2) A single clone of Pichia pastoris GS115 activated on a YPD plate was selected and cultured in 5ml of YPD liquid at 30 ℃ for 24h with shaking at 220rpm at constant temperature.

(3) A liquid transfer gun sucks 100uL of bacterial liquid to inoculate in 100mLYPD liquid, the temperature is 30 ℃, the rpm is 220, the time is 12-13h, the culture is carried out for 12h, the measurement is carried out at 0D600, and the 0D600 is strictly controlled at 1.3-1.5;

(4) observing whether there is contamination by microscopic examination, subpackaging thallus with 4 sterile 50ml centrifuge tubes, and standing on ice for 10-15 min;

(5) centrifugation was carried out at 1500 Xg, 4 ℃ for 5min, the supernatant was discarded, and each tube was used to resuspend the cells in 25mL of ice-cold sterile water;

(6) centrifuging at 4 deg.C for 5min at 1500 × g, and removing supernatant; resuspend the cells in 15mL (2/3) of ice-cold sterile water per tube;

(7) centrifuging at 4 deg.C for 5min at 1500 Xg, discarding supernatant, and resuspending thallus in 5ml of ice-cold 1M sorbitol per tube;

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

4. Yeast electric conversion

(1) After being dried, the electric cup is put into ice, the competent cells and the plasmids are put into the ice to be melted, and 1mL of sorbitol is taken for precooling.

(2) 100. mu.L of competence was mixed with 3-20. mu.g of the recovered linearized plasmid (less than 20. mu.L in volume) and transferred to a pre-cooled 0.2cm cuvette.

(3) The voltage is 2000v, the capacitance is 25VF and the resistance is 200 ohm.

(4) 600 and 700. mu.L of pre-cooled sorbitol were immediately added to the cup and mixed well and placed on ice for 5 min.

(5) Transferring the content of the electric rotating cup to a sterile 1.5mL EP tube in a super clean bench, standing in a thermostat at 30 ℃ for 1h

(6) The mixture was applied to a plate in 2 portions (plate pre-heated in an incubator at 30 ℃).

(7) Cultured at 30 ℃ until clone generation

5. Pichia pastoris culture and foreign protein induction expression

(A) The method comprises the following steps:

(1) single colonies on YPD plates were picked up with a 10. mu.L pipette tip and cultured in a conical flask containing 50mL of BMGY at 30 ℃ and 220rpm for 24 hours.

(2) Microscopic examination; measuring OD₆₀₀The value is obtained.

(3) Centrifuging at 24 deg.C for 10min at 3000g, collecting thallus, washing with BMMY once, and centrifuging. According to the OD value and the volume value measured in the last step, the bacterial concentration is diluted to 0D by BMMY₆₀₀＝1.0。

(4) The diluted bacterial solution was transferred to a 500mL conical flask and the yeast expression protein was induced at 29 ℃ and 220 rpm. Then methanol was added every 24h to a final concentration of 1% in the medium.

(5) The induction culture was continued for 84 h.

(6) And (5) microscopic examination, namely, the pH value of the fermentation liquor is required to be less than 6 when the condition that whether bacteria are infected exists is judged.

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

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

The results show (fig. 1): the expression results (lane 3) were in accordance with the requirements of further experiments, with a culture yield of 16 mg/L.

2.2 purification

Step A:

(1) the resulting supernatant of the fermentation broth was transferred to a conical flask, and NaCl was added so that the final concentration of NaCl was 150 mM.

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

(3)15000g, centrifuge for 30min to ensure that the supernatant is clear or transparent, otherwise the AKTA sampling tube is blocked.

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

(5) The sample was applied at a rate of 1mL/min, and the column-passing solution was collected while applying the sample, to prevent the loss of the protein not bound to the nickel column.

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

(7) The target protein was eluted with an Elution buffer (500mM imidazole), and one tube was collected every 5mL, approximately 20mL depending on the AU value.

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

As shown in FIG. 1 (lane 3), the purification effect was good and the bands were relatively uniform, and the concentrations of recombinant type XXI collagen were 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 conclusions

3.1 step

(1) Plate paving: cells in logarithmic growth phase were trypsinized to a single cell suspension and plated in 6-well plates, 3 wells per group; the cells were plated at 6 × 105 cells/well to ensure that the cells were confluent the next day, with a final total amount of 2 mL of medium per well;

(2) cell culture at 37 ℃ with 5% CO₂Culturing for 24h in an incubator;

(3) scratching: drawing a cross shape by using the gun head on the next day compared with a straight ruler, wherein the gun head is vertical (the same gun head is used among different holes);

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

(5) taking four fixed points of the cross shape, namely the upper point, the lower point, the left point and the right point of each hole, taking a picture under a 4-time lens, paying attention to the consistency of backgrounds, and sampling according to time points of 0h and 48 h;

(6) image data analysis

As can be seen from FIG. 2, the recombinant type XXI collagen at various concentrations significantly promoted migration of HaCaT cells after 48h of culture, in contrast to 0.28. mu. mol/L of COL21 acting similarly to 0.56. mu. mol/L.

Example 4: mouse skin injury repair experiment of recombinant collagen

4.1 construction of mouse full-thickness skin incision model

A full-thickness skin injury model is established by taking 24C 57BL/6 mice aged 7W, carrying out intraperitoneal injection anesthesia by using 5% L domethannin at a dose of 100 mu L, removing the hair on the back of the mice by using paraffin, printing a circular ring at the middle position of the back (preventing the mice from licking the wound) by using a skin drill with the thickness of 8mm, clamping the central skin of the circular ring by using forceps, and shearing the full-thickness skin along the circular ring by using a tissue shear to form a mechanical injury wound surface, wherein the day of model preparation is recorded as day 0. And after the molding is finished, carrying out single-cage feeding.

4.2 therapeutic intervention in mice

24 model mice were randomly divided into 3 groups of 8 mice each. The treatment is started on the day of modeling, a pipettor is used for sucking 120 mu L of liquid medicine respectively, the liquid medicine is slowly dripped on the wound surface, after the liquid medicine is completely absorbed, the mouse is placed back into the breeding cage for treatment for 2 times every day for 11 days continuously.

4.3 wound diameter healing Rate statistics

The size of the wound diameter was measured with a vernier caliper on days 1, 3, 5 and 7 of the molding, respectively, and the measurement was repeated three times for each mouse. The healing rate is (diameter of wound on the first day-diameter after treatment)/diameter of wound on the first day × 100%.

4.4 shoot to observe the wound healing

The recovery of the wound was recorded with a camera on days 1, 3, 5 and 7 of the model.

The experimental group was dosed at 0.4mg/mL (COL 21) and treated for 11 consecutive days with 240 μ l PBS (control) or treatment solution daily applied to the wound starting on the day of modeling. As can be seen from fig. 3 (fig. a), the wound defect areas of the control group and the experimental group were continuously reduced as the treatment days increased, but the wound area of the COL21 group was significantly reduced compared to the control group. When quantitative analysis is carried out on the diameter change of the wound (figures b and c), the healing speed of the COL21 group in the first 3 days is obviously higher than that of the control group (P <0.001), and no obvious difference exists in other time periods, and presumably, the concentration of the drug is too low, and the external drug is difficult to enter the deep part of the cortex along with the continuous epithelialization of the wound, so that higher concentration is needed to play the role.

The foregoing description is of the preferred embodiments only, which are by way of example only and do not limit 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 "including" are not intended to be limiting. Furthermore, unless otherwise indicated, the absence of a numerical modification includes the plural, and "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) Ltd

<120> recombinant human collagen and preparation method thereof

<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

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

595 600 605

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

625 630 635 640

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)

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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 (10)

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

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

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

4. Use of the recombinant human collagen of claim 1, the vector of claim 2, or the host cell of claim 3 for the preparation of a cosmetic, nutraceutical, or pharmaceutical agent.

5. The method for producing recombinant human collagen according to claim 1, comprising the steps of: (1) constructing a pichia pastoris gene engineering bacterium; (2) fermentation culture of pichia pastoris gene engineering bacteria; (3) inducing and expressing the recombinant human collagen; (4) and (5) purifying the recombinant human collagen.

6. The method of claim 5, wherein the pichia genetic engineering bacteria are constructed by the following steps:

(1) streak-culturing activated yeast GS115 on YPD plates;

(2) selecting a single clone of pichia pastoris GS115 activated on the YPD flat plate to be placed in the YPD liquid, and carrying out constant-temperature shaking culture;

(3) the liquid transfer gun sucks 100uL of bacterial liquid, inoculates the bacterial liquid in 100m LYPD liquid, the temperature is 30 ℃, the rpm is 220, 12-13h, and the culture is carried out until OD is reached₆₀₀＝1.3-1.5；

(4) Observing whether there is contamination by microscopic examination, subpackaging thallus with sterile 50ml centrifuge tube, and standing on ice for 10-15 min;

(5) centrifuging for 2 times, discarding supernatant, and resuspending thallus with sterile water;

(6) centrifuging for 2 times, discarding supernatant, resuspending thallus with sorbitol, and packaging to obtain competent cells;

(7) the vector is transferred into competent cells by an electric transfer method, and then the competent cells are subjected to plate culture until clone generation.

7. The method of any one of claims 5 or 6, wherein the fermentation culture step of the pichia pastoris genetically engineered bacteria is as follows:

(1) single colonies on YPD plates were picked and grown at 30 ℃ and 220rpm for 24h in a conical flask containing 50mL of BMGY, by measuring OD₆₀₀Confirming the end of the culture;

(2) centrifuging for 10min, collecting thallus, washing with BMMY, centrifuging, and diluting thallus concentration to OD₆₀₀＝1.0。

8. The method of any one of claims 5-7, wherein the steps of inducing and expressing the recombinant human collagen are as follows:

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

(2) microscopic examination, namely, observing whether the bacteria are infected or not, and measuring the pH value of the fermentation liquor, wherein the pH value is less than 6;

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

(4) taking out 1mL of supernatant, adding corresponding Loading buffer, mixing uniformly, incubating at 95 ℃ for 10min, and storing at-20 ℃.

9. The method of any one of claims 5-8, wherein the recombinant human collagen is purified by the following steps:

(1) transferring the obtained supernatant of the fermentation liquor into a conical flask, adding NaCl to ensure that the final concentration of the NaCl is 150mM, and adjusting the pH value of the supernatant of the fermentation liquor to 8.0 by adjusting NaOH or HCl;

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

(3) sampling at the speed of 1mL/min, and collecting the column-penetrating liquid while sampling;

(4) eluting unbound proteins and hetero-proteins with binding buffers of 5 nickel column bed volumes;

(5) eluting the target protein by using Elution buffer (500mM imidazole);

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

10. Recombinant human collagen obtained by the method according to any one of claims 5 to 9.

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