CN117866077B - Recombinant XVII type collagen and expression system thereof - Google Patents
Recombinant XVII type collagen and expression system thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
- A61K8/606—Nucleosides; Nucleotides; Nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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- General Health & Medical Sciences (AREA)
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Abstract
The invention provides recombinant XVII type collagen and an expression system thereof, and belongs to the technical field of development of cosmetic raw materials. According to the invention, through designing and splicing the sequence of the human XVII type collagen, two recombinant XVII type collagens are obtained, and the problem that the performance of the recombinant XVII type collagen in the prior art is to be improved is solved. The recombinant XVII type collagen provided by the invention has a better anti-aging and wrinkle-removing effect, and can play an excellent role at a low concentration. Experiments prove that the recombinant XVII type collagen provided by the invention has the effects of promoting skin cell proliferation and nourishing cell growth, and can promote collagen synthesis, so that the effects of resisting aging and removing wrinkles are achieved; the recombinant XVII type collagen can be absorbed into the dermis layer and has the effect of obviously increasing the thickness of the dermis layer, thereby achieving the effects of resisting aging, promoting the maintenance of skin functions and promoting the repair of tissue injury.
Description
Technical Field
The invention relates to the technical field of development of cosmetic raw materials, in particular to recombinant XVII type collagen and an expression system thereof.
Background
Transmembrane protein collagen XVII (COL 17/BP180/BPAG 2) is the structural component of the hemidesmosome (hemidesmosomes, HDs), the largest transmembrane collagen, a heterotrimer consisting of three 180kDa α1 (XVII) chains, each chain having a 466 amino acid intracellular globular N-terminal domain (ICD), a 23 amino acid short transmembrane region, and a 1008 amino acid extracellular C-terminal Ectodomain (ECD). The extracellular domain consists of 15 collagen subdomains (COL 1-COL 15), characterized by a typical collagen GXY repeat flanked by 16 short non-collagenous sequences (NC 1-NC 16A). COL17 is expressed primarily by basal keratinocytes, whose intracellular domain (ICD) binds to keratin via lectin (Plectin) and the like, while the Extracellular Domain (EDC) binds to integrin alpha 6 (integrin alpha 6), laminin-332 (laminin-332) and collagen IV (Col 4). These protein complexes form hemidesmosomes in the dermis-epidermis basement membrane zone, mediating adhesion of epithelial cells to the basal membrane.
Chinese patent CN116640205A discloses a recombinant XVII type collagen and an expression strain thereof, wherein the recombinant XVII type collagen sequence is obtained by combining functional domains combined with cytokinin in human XVII type collagen, and is respectively positioned in a collagen domain and a non-collagen domain of the human XVII type collagen, and comprises 2 non-collagen domains and 4 collagen domains in total, is transformed into pichia pastoris after codon optimization, and can stably express the recombinant XVII type collagen through high-copy Pichia pastoris genetic engineering bacteria obtained through G418 resistance gradient screening. The recombinant XVII type collagen expressed by the invention has excellent cell proliferation, migration and adhesion promoting performances of human hair follicle stem cells and human fibroblasts, can be used for repairing skin and hair follicle tissues, and improves the resistance of the skin and hair follicle.
However, the recombinant XVII type collagen in the prior art has to be improved in terms of anti-aging and wrinkle-removing properties.
Disclosure of Invention
The invention aims to provide recombinant XVII type collagen and an expression system thereof, which have better anti-aging and wrinkle-removing effects and can realize excellent effects at low concentration.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides recombinant XVII type collagen, and the amino acid sequence of the recombinant XVII type collagen is shown as SEQ ID NO. 1.
The invention also provides a gene for encoding the recombinant XVII type collagen, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
The invention provides another recombinant XVII type collagen, and the amino acid sequence of the recombinant XVII type collagen is shown as SEQ ID NO. 3.
The invention also provides a gene for encoding the recombinant XVII type collagen, and the nucleotide sequence of the gene is shown as SEQ ID NO. 4.
The invention also provides a recombinant vector for expressing the recombinant XVII type collagen, which comprises the initial vector and the gene for encoding the recombinant XVII type collagen.
The invention also provides a recombinant vector for expressing the recombinant XVII type collagen, which comprises the initial vector and the gene for encoding the recombinant XVII type collagen.
Preferably, the initial vector is a pPICZ alpha A vector, a pHIL-S1 vector, a pYAM P vector, a pPIC9 vector or a pPIC9K vector.
The invention also provides a recombinant strain for expressing recombinant XVII type collagen, wherein the recombinant strain is transformed with the recombinant vector.
Preferably, the recombinant strain expresses recombinant XVII type collagen by methanol induction.
The invention also provides application of the recombinant XVII type collagen, the recombinant vector and the recombinant strain in the production of skin repair products, wrinkle removal products or anti-aging products.
The invention has the beneficial effects that:
By designing and splicing the sequence of the human XVII type collagen, the invention obtains two recombinant XVII type collagens, has better action effect compared with the human XVII type collagen, can realize excellent effect with low concentration of protein, and has important significance for reducing industrialization difficulty. The cell viability experiment proves that the recombinant XVII type collagen has the effect of promoting the proliferation of skin cells and the growth of trophoblasts, and can especially promote the development and renewal of various cells in the human epidermis skin barrier system; cell experiments and related protein quantitative data show that the recombinant XVII type collagen can promote the synthesis of collagen, so that the effects of resisting aging and removing wrinkles are achieved; the human body experiment microneedle and Raman transdermal experiment show that the recombinant XVII type collagen can be absorbed into the dermis and has the effects of obviously increasing the thickness of the dermis, thereby achieving the effects of resisting aging, promoting maintenance of skin functions and promoting repair of tissue injury.
The expression system provided by the invention adopts pichia pastoris to recombinantly produce XVII type collagen, has the characteristics of high efficiency and low cost, and is suitable for popularization and application in actual industry.
Drawings
FIG. 1 is a graph showing the results of gel electrophoresis detection and analysis of proteins;
FIG. 2 is a diagram showing the proliferation of skin fibroblasts promoted by proteins;
FIG. 3 is a diagram showing the promotion of fibroblast synthesis of type I collagen (Col 1) by proteins;
FIG. 4 is a diagram showing the synthesis of collagen degrading metalloproteinase (MMP 1) by a protein-reduced fibroblast;
FIG. 5 is a graph showing the effect of protein on eliminating senescent cells;
FIG. 6 is a graph showing the results of the measurement of the duration of XVII type collagen in human skin after microneedle operation and the content of endogenous collagen in the skin;
FIG. 7 is a graph of the results of the protein human microneedle test for SS 001-numbered volunteers;
FIG. 8 is a graph of the results of the protein human microneedle test of SS 002-numbered volunteers;
FIG. 9 is a graph of the results of the protein human microneedle test for SS003 numbered volunteers;
FIG. 10 is a graph showing the results of the protein human microneedle test of the cheekbone of SS004 numbered volunteers;
fig. 11 is a graph of the results of the protein human microneedle test on the cheeks of SS004 numbered volunteers.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The invention is based on Col17 (XVII type collagen), and designs two recombinant proteins respectively named Col17-1 and Col17-2, and the sequence information is as follows:
Col17-1 amino acid sequence:
LNTNAYSAGSVFGVPNNMASCSPTLHPGLSTSSSVFGMQNNLAPSLTTLSHGTTTTSTAYGVKKNMPQSPAAVNTGVSTSAACTTSVQSDDLLHKDCKFLILEKDNTPAKKEMELLIMTKDSGKVFTASPASIAATSFSEDTLKKEKQAAYNADSGLKAEANGDLKTVSTKGKTTTADIHSYGSSGGGGSGGGGGVGGAGGGPWGPAPAWCPCGSCCSWWKWLLGLLLTWLLLLGLLFGLIALAEEVRKLKARVDELERIRRSILPYGDSMDRIEKDRLQGMAPAAGADLDKIGLHSDSQEELWMFVRKKLMMEQENGNLRGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMVDHHHHHH*, As shown in SEQ ID NO. 1;
Col17-1 nucleotide sequence:
TTGAATACTAACGCTTACTCAGCCGGCTCCGTTTTTGGTGTTCCAAACAACATGGCTTCCTGTTCTCCCACATTGCATCCAGGCTTGTCAACTTCTTCCAGTGTATTCGGAATGCAAAACAATTTGGCCCCTTCCCTGACCACTCTGTCACACGGTACTACTACCACCAGTACTGCCTATGGTGTAAAGAAAAACATGCCACAGTCACCCGCAGCAGTTAATACAGGTGTCTCCACTAGTGCTGCCTGTACCACATCTGTTCAATCTGACGACCTGTTGCATAAGGACTGTAAATTTCTGATCTTAGAGAAGGATAATACCCCAGCCAAAAAGGAGATGGAACTGTTGATAATGACCAAGGATTCAGGAAAGGTATTTACTGCCTCACCAGCATCCATTGCCGCTACTTCCTTCTCAGAGGATACGCTTAAGAAGGAAAAGCAGGCTGCCTATAACGCCGACTCTGGACTTAAGGCAGAAGCTAATGGCGATTTGAAGACTGTTAGTACAAAGGGTAAGACTACTACGGCCGACATTCACTCTTATGGTTCCTCAGGTGGTGGTGGTTCAGGAGGCGGAGGTGGTGTAGGTGGAGCAGGTGGAGGACCTTGGGGTCCAGCTCCTGCCTGGTGTCCTTGTGGTTCTTGCTGTTCTTGGTGGAAATGGTTATTGGGCCTGTTGTTAACCTGGTTATTGTTATTGGGTCTGTTGTTTGGTTTGATCGCTTTGGCAGAGGAAGTACGAAAATTGAAAGCCCGTGTTGACGAGTTGGAAAGAATCAGAAGATCCATATTGCCTTATGGAGACTCAATGGATAGGATTGAAAAAGACAGATTGCAGGGTATGGCTCCTGCCGCCGGTGCAGACCTGGACAAGATCGGACTGCACAGTGATTCTCAAGAGGAATTGTGGATGTTCGTTAGAAAGAAACTTATGATGGAACAAGAGAATGGTAACTTGAGAGGATCTCCCGGACCAAAAGGCGACATGGGATCTCCTGGTCCTAAGGGAGATAGAGGTTTTCCCGGTACTCCAGGAATCCCAGGTCCACTTGGTCACCCTGGTCCACAAGGCCCAAAGGGTCAAAAAGGATCAGTCGGTGATCCTGGAATGGAAGGCCCAATGGGTCAAAGAGGAAGGGAAGGACCAATGGGTCCCAGGGGAGAAGCTGGTCCACCAGGTTCTGGTGAAAAGGGCGAGAGAGGTGCAGCAGGTGAACCAGGTCCCCATGGACCTCCTGGTGTCCCCGGTTCAGTTGGACCAAAAGGTTCATCAGGTTCACCAGGTCCCCAGGGACCACCTGGACCAGTTGGATTGCAAGGATTACGAGGCGAGGTTGGATTACCCGGTGTAAAGGGTGACAAGGGTCCTATGGGACCCCCTGGTCCAAAGGGCGATCAGGGTGAGAAGGGCCCAAGAGGTCTAACGGGAGAACCCGGCATGCGAGGACTACCCGGTGCTGTCGGTGAGCCTGGAGCTAAAGGTGCCATGGGTCCAGCCGGTCCTGATGGTCACCAAGGTCCTCGTGGAGAACAAGGTTTGACAGGTATGCCCGGTATCAGAGGACCTCCTGGTCCTAGTGGAGATCCTGGAAAGCCTGGACTTACCGGTCCACAGGGCCCTCAAGGACTACCAGGTACTCCAGGTCGTCCTGGTATAAAAGGAGAACCTGGTGCCCCTGGTAAAATAGTGACCTCCGAGGGAAGTTCTATGGTCGACCATCATCATCATCATCATTGA, As shown in SEQ ID NO. 2;
col17-2 amino acid sequence:
LQGPPGPPGPQGPKGDKGDPGVPGALGIPSGPSEGGSSSTMYVSGPPGPPGPPGPPGSISSSGQEIQQYISEYMQSDSIRSYLSGVQGPPGPPGPPGPVTTITGETFDYSELASHVVSYLRTSGYGVSLFSSSISSEDILAVLQRDDVRQYLRQYLMGPRGPPGPPGASGDGSLLSLDRGSSYSSSMSTGGGGAGSLGAGGAFGEAAGDRGPYGTDIGPGGGYGAAAEGGMYAGNGGLLGADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQGPPGQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQVYAGRRRRRSIAVKPVDHHHHHH*, As shown in SEQ ID NO. 3;
col17-2 nucleotide sequence:
TTGCAAGGACCACCAGGCCCACCTGGACCTCAAGGACCAAAGGGTGACAAAGGAGATCCAGGTGTTCCTGGAGCTTTGGGTATCCCATCAGGCCCCAGTGAAGGCGGTTCATCATCTACCATGTACGTCAGTGGACCACCTGGACCTCCCGGACCCCCAGGACCACCTGGTTCCATTTCTTCTTCTGGACAGGAAATTCAGCAGTACATTTCCGAGTACATGCAATCCGATTCAATTCGTTCATACTTGTCCGGTGTTCAGGGTCCTCCAGGTCCTCCCGGTCCTCCCGGACCTGTTACTACTATAACTGGTGAAACTTTCGATTACAGTGAGTTGGCCTCCCATGTTGTTTCTTACCTGCGTACATCCGGATATGGTGTGTCTCTGTTCTCAAGTTCCATCTCATCCGAGGACATCCTGGCTGTGCTACAGAGGGACGACGTTAGGCAGTATTTAAGACAATATCTAATGGGTCCAAGGGGTCCACCTGGTCCACCAGGCGCCTCAGGTGATGGATCTTTGTTGTCATTGGATCGTGGATCTTCATATTCCTCTTCCATGTCAACAGGAGGAGGAGGCGCAGGTTCATTGGGAGCAGGAGGAGCATTTGGTGAAGCCGCAGGTGACCGAGGACCTTATGGTACTGACATCGGACCTGGTGGTGGTTATGGAGCTGCTGCTGAGGGAGGTATGTATGCAGGTAATGGAGGTTTACTAGGCGCAGACTTCGCCGGCGACCTGGATTATAATGAGCTTGCCGTCAGAGTTAGTGAGTCTATGCAACGTCAAGGACTTCTGCAGGGAATGGCTTACACGGTACAAGGTCCACCAGGACAACCTGGACCCCAAGGACCACCAGGAATTTCAAAGGTGTTTTCAGCATATAGTAACGTCACGGCTGATCTGATGGATTTCTTCCAGACCTATGGTGCTATCCAAGGACCTCCAGGTCAAAAAGGTGAAATGGGTACCCCAGGTCCTAAGGGTGACAGAGGTCCAGCTGGACCTCCAGGTCATCCAGGTCCACCAGGTCCAAGAGGCCATAAGGGTGAAAAGGGAGATAAGGGCGACCAGGTGTACGCAGGAAGAAGGAGGCGTAGGTCCATCGCCGTGAAGCCTGTCGACCATCATCATCATCATCATTGA, As shown in SEQ ID NO. 4;
The nucleotide sequences are respectively connected to obtain recombinant expression vectors pPICZ alpha A-Col17-1 and pPICZ alpha A-Col17-2, the two recombinant proteins are respectively connected with a secretion signal peptide (alpha-factor) carried by a tandem vector by taking AOX induced by methanol as a promoter, and the C terminal is fused with a histidine tag.
Pichia pastoris expression vectors were then linearized using SacI. This procedure was followed by cleavage of 5. Mu.g of plasmid with SacI endonuclease in a 100. Mu.L system according to the instructions given for NEB. The plasmid after cleavage was run on a DNA gel and recovered with the kit. Single colonies were picked from the non-resistant YPD-X33 plates and inoculated into shake flasks containing 5 mL YPD (tryptone (peptone) 20g/L, yeast powder (Yeast extract) 1g/L, naCl g/L) and shaken overnight at 30℃220r to an OD600 of approximately 2-6 (approximately 16-18h was required). The cell concentration was observed, 1% was inoculated into 200 mL YPD medium, and cultured at 30℃220r until the OD600 was 1.3-1.5 (about 6 hours was required). 4. Centrifugation at 1500 r C min was performed, and after the cells were collected, the cells were resuspended in 50 mL ice pre-chilled sterile water and repeated twice. After the cells were collected, the cells were resuspended in 25 mL ice pre-chilled 1M sorbitol solution and repeated twice. The cells were collected by centrifugation and resuspended in 200. Mu.L of ice pre-chilled 1M sorbitol solution. After adding the current plasmid and mixing, transfer into an electric rotating cup which is cooled, and place on ice for 5min. 1mL of ice pre-chilled 1M sorbitol solution is added immediately after electric shock is performed according to parameters of voltage 1.5 Kv, capacitance 25 mu F, resistance 200 omega and electric shock time of 4-10 msec. After incubation for 1h at 30℃and 220rpm, 400. Mu.L of the bacterial liquid was plated (0.1 mg/ml zeo) and incubated in an incubator at 30℃until single colonies were grown. All single colonies were picked to 0.1mg/ml zeo resistance plates for colony purification and transferred to 2.0. 2.0 mg/ml zeo resistance plates the next day for pressure screening of high copy colonies.
2 Mg/mL bleomycin plates were observed, transformants with slightly better growth vigor were selected and inoculated into 3 mL YPD medium (10 mL tube) respectively, and cultured overnight at 30℃220 r; taking 500 mu L of the bacterial liquid (1%) and inoculating the bacterial liquid into 50ml of BMGY culture medium (250 ml of culture flask), and shaking the bacterial liquid at the temperature of 30 ℃ 220 r until the OD 600 is 2-6;3000 r centrifugation 10 min, after suspension with appropriate amount of BMMY medium, transfer to 200mL BMMY medium (1000 mL flask) to OD 1.0; sampling every 24h times, adding methanol to a final concentration of 0.5%, and performing SDS-page identification expression on the extracted sample to identify Pichia pastoris with good expression effect, and performing amplification culture to prepare the recombinant XVII type collagen.
Centrifuging the culture solution at 10000rpm for 10min, and collecting the supernatant; passing through a 0.45 μm membrane. The Ni column was equilibrated by washing 5 volumes of equilibration liquid (40 mM NaH 2PO4, 40mM Tris, supplemented with ultra pure water to 950 mL, after complete dissolution, HCI was adjusted to pH 7.0, and finally to 1L, stored at room temperature) at a flow rate of 10ml/min. Loading the culture supernatant onto column, collecting the flow-through liquid, and repeating the loading process for 2-3 times. The column was washed with 5 column volumes of equilibration solution to wash off the contaminating proteins and the effluent was collected. The column was washed with 5 column volumes of washing solution (40 mM NaH 2PO4, 40mM Tris,150mM imidazole, supplemented with ultra pure water to 950 mL, after complete dissolution, HCI was adjusted to pH 7.0, finally to 1L, stored at room temperature) to wash out the contaminating proteins, and the effluent was collected. Eluting the column with 5 times of column volume eluent (40 mM NaH 2PO4, 40mM Tris,300mM imidazole, adding ultrapure water to 950 mL, completely dissolving, adjusting pH to 7 with HCl, finally fixing volume to 1L, and preserving at room temperature), and collecting effluent to obtain purified target protein solution.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodi. Mu. m dodecyl s. Mu. lfatepolyacrylamide gel electrophoresis, SDS-PAGE) analysis showed that the molecular weight of the proteins was approximately 58.2 and 37.2 kilodaltons (kDa), as shown in FIG. 1.
And freeze-drying the purified protein by a low-temperature vacuum drying method to obtain recombinant protein freeze-dried powder and storing.
Example 2 fibroblast Activity assay
Skin primary fibroblasts were inoculated into modified eagle medium (Dulbecco's Modified Eagle Medium, DMEM), cultured at 37 ℃ to logarithmic growth phase, and then inoculated into 96-well cell culture plates (5000 cells/well, 100 μl medium/well). Protein lyophilized powder was diluted to 1mg/mL with phosphate buffer (137 mM sodium chloride, 2.7mM potassium chloride, 10mM sodium dihydrogen phosphate, 2mM potassium dihydrogen phosphate, 10EU thrombin, 1mM calcium chloride and 0.5mM magnesium chloride, pH=7.2), and then added to 96-well cell culture plates in proportion so that the final concentration of recombinant XVII-type collagen was sequentially 1ppm, 5ppm, 10ppm, 50ppm and 100ppm, with the samples to which phosphate buffer was added as a blank. 3 replicate wells were treated for each concentration. After 48 hours of incubation at 37 ℃, a final concentration of 5 mg/ml of thiazole blue solution (MTT, 10 μl/well) was added and incubation was continued for 4 hours at 37 ℃. The medium was discarded, 150. Mu.L of dimethylmethylene cut (DMSO) was added to each well, and then placed on a shaker for slow shaking for 10 minutes. Finally, the absorbance of each well was measured by an enzyme-labeled instrument at a wavelength of 490nm, and the results are shown in FIG. 2.
The results showed that the cell viability of the experimental group to which both proteins were added was greater than 100% compared to the control group (HACAT, CT 1), both promoting fibroblast viability. The Col17-1 protein has 114% cell survival rate only at 1ppm concentration, and has cell survival rate of more than 120% at 50ppm concentration. The cell survival rate of the Col17-2 protein can reach more than 120% at the concentration of 100ppm, and the result shows that the recombinant XVII type collagen has obvious proliferation promoting effect on skin fibroblasts.
EXAMPLE 3 quantitative analysis of the effects of recombinant XVII-type collagen on collagen-related proteins
Cells in logarithmic growth phase are inoculated into 6-well plates by 6x10 x 5 skin primary fibroblast cells per well, after being cultured for 24 hours (confluence of 80% -90%) in a constant temperature incubator with 37 ℃ and 5% CO 2 and 95% humidity, the culture medium is discarded, the culture medium is washed 2 times with PBS, the residual culture medium is washed away, 2mL of detection sample with the final concentration of detection concentration, which is obtained by diluting with serum-free culture medium (which can contain 1% diabody), is added into each well, and serum-free (which can contain 1% diabody) culture medium is used as a control group. Put into a 37 ℃ and 5% CO2 incubator, and sample 24 hours after administration. Each experimental sample Kong Xian was washed 2-3 times with PBS, after sucking clean liquid, 0.5ml Trizol was added to each well, repeated blow-sucking was performed, adherent cells were thoroughly blown down, and the samples were all transferred to 1.5ml centrifuge tubes without RNase. RNA was extracted using the kit, and the detailed procedure is referred to the kit instructions. Reverse transcription of cDNA, for details, reference is made to reverse transcription instructions. The three-step method qPCR (TAKARA TB Green Premix Ex Taq (TLI RNASEH Plus), detailed steps refer to qPCR reagent specifications, statistical analysis software (excel, graphpad, spss, etc.) is used for calculating the mean value (mean) of each detection sample Ct first, then the Ct of the detection sample, that is, the Ct of a certain detection factor of a certain detection sample, that of a certain detection sample of an internal gene of a certain detection sample, then the Ct of the detection sample, that is, the Ct of a certain detection factor experimental group, that of a certain detection factor blank group, and finally the Ct of the detection sample, that is, the relative expression amount, and the standard error (SD) of each detection sample hole are calculated, and data tables are obtained, and the results are shown in FIG. 3 and FIG. 4.
The results show that the addition of recombinant XVII type collagen can significantly improve the synthesis of type 1 collagen, wherein Col17-1 can improve 62% of collagen synthesis at 5ppm, and Col17-2 can improve 45% of collagen synthesis at 50 ppm. It can also be seen from the quantitative result of transcription of metalloproteinase MMP1 that when 5ppm of Col17-1 is added, the transcription level of MMP1 is only 32%, and 50ppm is only 22%. The transcript levels of MMP1 were also significantly reduced in the experimental group with Col17-2 added.
Therefore, the recombinant XVII type collagen provided by the invention can effectively increase the synthesis of the I type collagen in fibroblasts and reduce the synthesis of metalloproteinase MMP1, thereby achieving the effects of increasing the content of collagen and improving the elastic support of skin.
EXAMPLE 4 Effect of scavenging senescent cells
The logarithmic phase of fibroblasts was seeded at 1×10 6 cells/well in 6-well plates and incubated in a 5% CO 2 incubator at 37 ℃ for 24 h. The culture solution was discarded, and after HaCaT cells were irradiated with UVB 90mJ/cm 2 (excluding the blank group), experimental samples (Col 17-1 and Col17-2 were prepared at final concentrations of 5ppm and 50ppm, respectively) and serum-free medium (model group, blank group) were added; the above samples were used in subsequent experiments after incubation in a 37℃5% CO 2 incubator for 24: 24 h. The cell culture broth was aspirated, washed 1 time with PBS, and 1 ml of beta-galactosidase staining fixative was added and fixed at room temperature for 30 minutes. The cell fixative was aspirated and the cells were washed 3 times for 3 minutes with PBS. The PBS was removed by pipetting, and 1 ml of staining fluid was added to each well. The preparation method of the dyeing working solution is referred to in the following table 1. Incubate overnight at 37 ℃, seal the 6-well plate edge with sealing film and fit into a seal pocket to prevent evaporation. Note that: incubation at 37℃cannot be performed in a carbon dioxide incubator. The aged cells were observed under a normal light microscope and photographed for their ratio, and the results are shown in FIG. 5.
TABLE 1 preparation method of dyeing working solution
The results show a significantly effective reduction in senescent cells in the 5ppm Col17-1 experimental group compared to the model group, with similar effects for Col 17-2. Also, compared to the blank, senescent cells were even fewer in the experimental group with the addition of recombinant XVII type collagen.
Therefore, the recombinant XVII type collagen provided by the invention can effectively remove aged cells and maintain the healthy environment of skin cells.
Example 5 human microneedle transdermal verification
The subjects cleaned the face with facial cleanser, then applied with the anesthetic (2% lidocaine) for 15min, removed the dressing, applied with 10mg/ml Col17-1 collagen dressing (formulation of panthenol 12.5mg/ml, col17-1 collagen 10mg/ml,1, 2-pentanediol 5mg/ml,1, 3-propanediol 6mg/ml, sodium hyaluronate 2mg/ml, aqueous solution), and then rolled back and forth 5 times on the skin surface with 1.0 mm roller. D1 is the detection result of the second day after operation, the rest days and so on.
The LabRAM Odyssey high-speed high-resolution microscopic confocal Raman spectrometer is used for testing, the parameters are 1800 (500 nm) grating scale, 532nm-Edge light source, 0.1% -50% light source intensity and X50-vis microscope. And (3) finding out the skin surface of the human body by adjusting the focal length in the bright field, switching to a laser light source, and adjusting the focal length until the green light spots displayed on the screen are minimum. Performing baseline calibration and characteristic peak position confirmation on a Raman spectrum by adopting Labspec software in data analysis, and calculating the obtained Raman spectrum, wherein the obtained Raman spectrum comprises peak intensity, peak displacement, peak area, half-peak width and the like; meanwhile, labspec software is used for carrying out numerical analysis of peak intensities corresponding to different depths and drawing of spatial distribution of the peak intensities, and the more red the color is, the higher the concentration of a target test object is. The concentration of XVII type collagen and the endogenous collagen content in the skin after the microneedle were measured in this experiment, and the results are shown in FIG. 6.
The results show that a large amount of XVII type collagen can be obviously measured after the microneedle operation, and the XVII type collagen still exists on the 14 th day after the operation, so that the XVII type collagen can effectively enter epidermis and dermis layers to act. Meanwhile, from the detection of the endogenous collagen content of the skin, the content of the collagen in the dermis layer and the active epidermis layer is obviously increased after the microneedle operation. Therefore, the recombinant XVII type collagen can promote the synthesis of collagen and improve the content of collagen in human skin.
Example 6 human microneedle skin dermis layer thickness test
Microneedle surgery was performed as in example 5 by ultrasonic imaging of the skin by means of an instrument Ultrascan UC 22. The device obtains information such as skin thickness, collagen density in the skin and the like through skin ultrasonic imaging, and outputs an image. After 3 weeks of surgery on the face, 4 subjects were tested on the cheeks and cheeks, respectively, and the results are shown in fig. 7.
The results showed that the zygomatic dermis densities of the subjects SS001 and SS004 were significantly increased and the zygomatic dermis thicknesses of the subjects SS001, SS002 and SS004 were significantly increased after 3 weeks of the microneedle operation. The cheek dermis density and dermis thickness of the subjects SS003 and SS004 are both significantly improved.
Therefore, the recombinant XVII type collagen provided by the invention can effectively improve the thickness and density of the dermis layer, thereby achieving the anti-aging effect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The recombinant XVII type collagen is characterized in that the amino acid sequence of the recombinant XVII type collagen is shown as SEQ ID NO. 1.
2. A gene encoding recombinant XVII type collagen according to claim 1, wherein the nucleotide sequence of said gene is shown in SEQ ID No. 2.
3. The recombinant XVII type collagen is characterized in that the amino acid sequence of the recombinant XVII type collagen is shown as SEQ ID NO. 3.
4. A gene encoding recombinant XVII type collagen according to claim 3, wherein the nucleotide sequence of said gene is shown in SEQ ID No. 4.
5. A recombinant vector for expressing recombinant XVII type collagen, wherein said recombinant vector comprises a starting vector and the gene of claim 2.
6. A recombinant vector for expressing recombinant XVII type collagen, wherein said recombinant vector comprises a starting vector and the gene of claim 4.
7. The recombinant vector according to claim 5 or 6, wherein the initial vector is a ppiczαa vector, a pHIL-S1 vector, a pYAM P vector, a pPIC9 vector or a pPIC9K vector.
8. A recombinant strain expressing recombinant XVII type collagen, wherein the recombinant strain is transformed with the recombinant vector of any one of claims 5 to 7.
9. The recombinant strain of claim 8, wherein the recombinant strain expresses recombinant XVII type collagen by methanol induction.
10. Use of the recombinant XVII collagen according to claim 1 or 3, the recombinant vector according to any one of claims 5 to 7 or the recombinant strain according to claim 8 or 9 for the production of skin repair products, wrinkle removal products or anti-aging products.
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