CN118324899B - Recombinant XVII type humanized collagen, preparation method and application thereof - Google Patents
Recombinant XVII type humanized collagen, preparation method and application thereof Download PDFInfo
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Abstract
The application discloses recombinant XVII type humanized collagen, a preparation method and application thereof, and belongs to the technical field of collagen and genetic engineering. The amino acid sequence of the recombinant XVII type humanized collagen prepared by the application is shown as SEQ ID NO. 1, a whole set of fermentation process and purification process are established, the protein expression level is improved, NO degradation exists, and experiments prove that the obtained protein has better biological activities such as cell adhesion, proliferation performance and the like on the commercial XVII type collagen, can be developed into a new functional biological raw material, and is widely applied to the fields of pharmaceutical compositions, medical equipment, tissue engineering products or cosmetics and the like.
Description
Technical Field
The invention relates to the technical field of collagen and genetic engineering, in particular to recombinant XVII type humanized collagen, a preparation method and application thereof.
Background
Collagen is one of the most important and abundant proteins in mammals, a structural protein found in human skin, connective tissue and bone, and other tissues. Collagen is generally white, transparent and unbranched fibril, is a basic support for skin and bones, can account for 25% -35% of the total amount of protein, is mainly distributed at the skin, blood vessels, bones, tendons, teeth, cartilage and other parts of a human body, is a main matrix and a bracket of the tissues, protects and connects various tissues, and plays an important physiological function in the body. Therefore, the collagen can be widely applied to industries such as medicines, cosmetics and the like.
The human body contains 28 different types of collagens, which are classified into common fibrous collagens and unusual non-fibrous collagens. Type I, type II and the like in human skin belong to fiber collagen. Among the non-fibrillar collagens, there is a very important collagen subtype, collagen type 17 (XVII type collagen, COL17, type 17 collagen, BP 180), which is a transmembrane non-fibrillar collagen, which is a homogeneous trimer of three identical α1 (XVII) chains, with a single chain molecular weight of up to 180kDa. The human XVII type collagen is a component of hemidesmosome in cells, plays an important role in the action of epithelial cell basement membrane, can regulate and control the adhesion, separation and development and differentiation of epithelial cells, and has important roles in cell aging and skin differentiation.
The human XVII type collagen has very little content in human body, the XVII type collagen has very little content in animal body, the extraction difficulty is very high, the mass production cannot be realized, and meanwhile, the animal-derived collagen product inevitably has immunogenicity and potential biological safety hazards such as viruses, epidemic diseases and the like, and has no mass application. Limited to ethical constraints, human XVII type collagen can only be applied to scientific research, and the commercial XVII type collagen products of human or other animal sources are not available in the market, so that the research and application of human XVII type collagen are further limited, and the development of an efficient green manufacturing platform for mass production of recombinant XVII type collagen has important significance for promoting the application of rare collagen in the fields of daily chemicals, skin care, health care, medical treatment and the like. The main way to solve such problems is to complete collagen by biotechnology such as genetic engineering.
With the rapid development of genetic engineering and synthetic biology, the construction of artificial design and construction of biological systems has become a mainstream trend of biology nowadays, and the de novo design of genetic element modules is the basis of synthetic biology. Based on the concept, by combining with a protein engineering rational transformation strategy, the recombinant XVII type collagen with high biological performance is reasonably designed, and the efficient expression of the recombinant XVII type collagen in a heterologous host is realized, so that the application of the XVII type collagen in industrial production is greatly facilitated, and the construction of the genetic engineering strain by utilizing a genetic engineering technology means has important significance for preparing high-quality human-like collagen.
Disclosure of Invention
The technical problem to be solved by the invention is to use a genetic engineering technology to recombinant express a novel recombinant XVII type humanized collagen, and a preparation method and application of the recombinant XVII type humanized collagen.
Based on the above, the technical scheme of the invention is realized as follows:
the invention provides recombinant XVII type humanized collagen, and the amino acid sequence of the collagen is shown as SEQ ID NO. 1.
The invention also provides a nucleic acid molecule for encoding the recombinant XVII type humanized collagen.
Further, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.
The invention also provides a recombinant expression vector comprising the nucleic acid molecule.
The invention also provides a host cell comprising the recombinant expression vector or expressing the collagen.
Further, the host cell is E.coli BL21 (DE 3).
By host cell is meant any cell type that is susceptible to transformation, transfection, transduction, etc. by a nucleic acid construct or expression vector comprising a polynucleotide of the application. The host cell may be any cell useful in the production of recombinant humanized collagen of the present application. To produce recombinant collagen, nucleic acid encoding recombinant collagen may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding recombinant collagen). The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be identical in nucleic acid content to the parent cell, but may contain mutations. Methods for introducing vectors into host cells are well known, for example, using electrotransformation to introduce vectors into host cells, and may also be transfection, microinjection techniques, gene gun techniques, liposome-mediated methods, and the like. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from any one of pichia pastoris, saccharomyces cerevisiae, escherichia coli and bacillus subtilis. Preferably, the prokaryotic cell is escherichia coli; preferably, the eukaryotic cell is pichia pastoris.
The invention also provides a preparation method of the recombinant XVII type humanized collagen, which is characterized by comprising the following steps:
(1) Culturing said host cell in a culture medium;
(2) Isolating the XVII type humanized collagen from the host cell.
The invention also provides a composition comprising the recombinant XVII-type humanized collagen, or the recombinant XVII-type humanized collagen encoded by the nucleic acid molecule, or the humanized XVII-type collagen expressed by the recombinant expression vector, or the recombinant XVII-type humanized collagen produced by the host cell, or the recombinant XVII-type humanized collagen produced by the production method.
The invention also provides an article comprising the composition, which is a pharmaceutical composition, a medical device, a tissue engineering product or a cosmetic.
Further, the product is one or more of biological dressing, human bionic material, plastic and beauty material, organoid culture material, cardiovascular stent material, coating material, tissue injection filling material, ophthalmic material, obstetrical and gynecological biological material, nerve repair regeneration material, liver tissue material and blood vessel repair regeneration material, 3D printing artificial organ biological material, cosmetic raw material, pharmaceutical auxiliary material and food additive.
Further, the portion of the cosmetic is not particularly limited, and may be a face, a hand, a leg, a trunk, or the like. The form of the composition is not particularly limited as long as the intended function can be achieved. For example, the composition is a solid, liquid or gel composition.
The invention also provides application of the composition in preparing a pharmaceutical composition, a medical device, a tissue engineering product or a cosmetic.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a novel recombinant XVII type humanized collagen SEQ ID NO. 1, a section of humanized XVII type collagen gene is designed based on a collagen characteristic sequence Gly-X-Y, and amplified and cloned into a pPIC9K vector after codon optimization, and then converted into an escherichia coli cell for expression after enzyme digestion linearization to obtain a target protein. The invention establishes a whole set of fermentation process and purification process, the prepared XVII type humanized collagen has good hydrophilicity and higher protein expression level and stability, and experiments prove that the obtained protein has better biological activities such as cell adhesion, proliferation performance and the like for the commercial XVII type collagen, can be developed into a new functional biological raw material, and is widely applied to the fields of pharmaceutical compositions, medical equipment, tissue engineering products or cosmetics and the like.
Drawings
FIG. 1 is a SDS-PAGE result of recombinant XVII-type humanized collagen expression, having an apparent molecular weight of about 50.55kDa. Wherein: lane 1 is a sample of the supernatant from step (3) of example 1, and lane 2 is a sample of purified recombinant XVII-type humanized collagen.
FIG. 2 is an SEM image of recombinant XVII-type humanized collagen.
FIG. 3 is an external view of recombinant XVII-type humanized collagen.
FIG. 4 shows cytotoxicity of recombinant XVII-type humanized collagen.
FIG. 5 shows the cell proliferation promoting activity of recombinant XVII-type humanized collagen.
FIG. 6 shows the cell adhesion promoting activity of recombinant XVII-type humanized collagen.
Detailed Description
The present invention will be described below by way of examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the medicinal materials and the reagents are obtained from commercial sources unless otherwise specified; and the properties of products from different sources have no significant effect.
The features, advantages and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure.
All percentages, fractions and ratios are calculated on the total mass of the composition of the invention, unless otherwise indicated. The term "mass content" is used herein to denote the symbol "%".
The terms "comprising," "including," "containing," "having," or other variations thereof herein are intended to cover a non-closed inclusion, without distinguishing between them. The term "comprising" means that other steps and ingredients may be added that do not affect the end result. The term "comprising" also includes the terms "consisting of and" consisting essentially of. The compositions and methods/processes of the present invention can comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as additional or optional ingredients, components, steps, or limitations of any of the embodiments described herein.
Example 1 Gene design and Synthesis
And (3) screening the large-scale functional region to obtain the target gene functional region of the recombinant XVII type humanized collagen COL 17-HR. The amino acid sequence of COL17-HR is shown as SEQ ID NO. 1:
GPRGAPGKRGSQGPKGDMGSPGPKGDRGFPGTPGIPGILGHPGPQGPKGQKGSVGDPGMEGPGEPGLQGLRGLVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGAPGMRGLPGAVGEPGTPGIPGPLGHPGPQGPKGQKGPVGDPGMEGPMGQRGREGKMGPRGEAGPPGSGDRGAPGPRGAPGKRGSQGPKGDMGSPGPKGDRGFPGTPGIPGILGHPGPQGPKGQKGSVGDPGMEGPGEPGLQGLRGLVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGAPGMRGLPGAVGEPGTPGIPGPLGHPGPQGPKGQKGPVGDPGMEGPMGQRGREGKMGPRGEAGPPGSGDRGAPGPRGAPGKRGSQGPKGDMGSPGPKGDRGFPGTPGIPGILGHPGPQGPKGQKGSVGDPGMEGPGEPGLQGLRGLVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGAPGMRGLPGAVGEPGTPGIPGPLGHPGPQGPKGQKGPVGDPGMEGPMGQRGREGKMGPRGEAGPPGSGDRGAP.
The gene sequence is reversely designed by using an online codon optimization tool (ExpOptimizer) (https:// www.novopro.cn/tools/codon-optimization. Html), and EcoRI and NotI restriction sites are removed in the design process aiming at preferred codons required by the expression of host escherichia coli, so that the gene sequence after optimization is shown as SEQ ID NO. 2, the GC content after optimization is reduced to 61.80%, and the gene is more beneficial to the high-efficiency expression of the host escherichia coli.
The gene sequence after COL17-HR optimization is shown as SEQ ID NO. 2:
GGTCCACGTGGTGCGCCGGGTAAACGTGGCTCTCAAGGCCCAAAAGGTGATATGGGTTCTCCGGGTCCGAAAGGTGATCGCGGCTTTCCGGGTACTCCGGGCATTCCGGGTATCCTGGGTCACCCGGGTCCTCAGGGCCCAAAAGGCCAGAAAGGCTCCGTTGGTGATCCAGGCATGGAAGGTCCGGGTGAACCTGGTCTGCAAGGTCTGCGTGGTCTGGTAGGCCTGCCTGGTGTTAAAGGTGACAAAGGCCCTATGGGCCCACCTGGCCCAAAAGGTGACCAGGGTGAAAAAGGTCCGCGTGGTCTGACTGGTGCTCCAGGTATGCGTGGCCTGCCGGGTGCAGTTGGCGAACCAGGTACGCCTGGTATTCCGGGTCCACTGGGTCACCCTGGTCCGCAAGGTCCGAAAGGTCAGAAGGGTCCTGTAGGCGACCCGGGTATGGAGGGTCCAATGGGTCAGCGTGGTCGCGAAGGTAAAATGGGTCCACGTGGTGAAGCTGGTCCGCCTGGTAGCGGTGATCGTGGTGCACCTGGTCCGCGTGGTGCACCAGGTAAGCGTGGTAGCCAGGGTCCGAAAGGCGACATGGGTAGCCCGGGTCCGAAGGGTGATCGTGGTTTCCCGGGTACCCCGGGTATCCCGGGTATTCTGGGTCACCCAGGTCCTCAGGGTCCGAAAGGCCAAAAAGGTTCTGTAGGTGATCCAGGTATGGAAGGCCCGGGTGAACCAGGTCTGCAGGGTCTGCGCGGTCTGGTTGGTCTGCCAGGTGTGAAAGGTGATAAAGGCCCGATGGGCCCGCCAGGTCCAAAAGGTGATCAAGGTGAAAAAGGTCCTCGTGGTCTGACGGGTGCCCCAGGTATGCGTGGTCTGCCGGGTGCTGTTGGCGAACCAGGTACTCCTGGTATTCCTGGTCCTCTGGGTCATCCAGGCCCTCAGGGTCCGAAAGGTCAGAAAGGCCCGGTTGGTGACCCGGGCATGGAAGGTCCGATGGGTCAGCGTGGTCGTGAAGGCAAAATGGGTCCGCGTGGTGAAGCTGGCCCACCAGGTTCTGGTGATCGTGGTGCACCGGGTCCTCGTGGTGCACCTGGTAAACGCGGCTCCCAGGGTCCTAAAGGCGATATGGGCTCTCCGGGTCCTAAGGGTGATCGTGGTTTTCCAGGCACCCCAGGTATCCCTGGCATTCTGGGCCATCCGGGTCCACAGGGCCCGAAAGGTCAGAAAGGTTCTGTTGGCGATCCGGGCATGGAAGGTCCAGGTGAACCGGGTCTGCAGGGTCTGCGTGGTCTGGTTGGTCTGCCGGGTGTTAAAGGTGATAAAGGTCCGATGGGTCCACCGGGCCCGAAAGGTGATCAGGGCGAAAAGGGCCCTCGTGGTCTGACTGGTGCACCAGGCATGCGTGGCCTGCCAGGTGCAGTTGGTGAACCTGGTACCCCGGGCATTCCAGGTCCGCTGGGTCATCCTGGTCCGCAGGGTCCTAAGGGTCAGAAAGGTCCGGTGGGTGACCCTGGCATGGAAGGTCCTATGGGTCAACGTGGCCGCGAGGGCAAAATGGGTCCTCGTGGTGAAGCAGGTCCGCCAGGTTCTGGTGACCGTGGTGCTCCG.
The nucleotide fragment of 1602 bases according to the gene sequence shown in SEQ ID NO. 2 was submitted to total gene synthesis by the division of biological engineering (Shanghai).
EXAMPLE 2 construction, expression and purification of vectors
(1) Cloning and amplifying the DNA fragment of SEQ ID NO. 2 by using a PCR method, and recovering the target gene by agarose gel electrophoresis. Cutting the gene fragment into cohesive ends by using the enzyme cutting sites of EcoRI and NotI added on the primers, adding corresponding restriction enzymes, incubating with a pPIC9K expression vector (purchased from Simer Feishr technology Co.) subjected to corresponding enzyme cutting treatment, adding T4 DNA ligase (TaKaRa Co., product No. 2011A), and connecting at 4 ℃ for 12 hours to convert escherichia coli DH5 alpha (purchased from Shanghai) of biological engineering (Shanghai); screening white spot clones by using an ampicillin sodium-LB plate, extracting plasmids by using an alkaline lysis method or a boiling method of molecular cloning, identifying positive clones to be correct by using a method of enzyme digestion and PCR, finally sequencing to determine that the recombinant plasmids contain correct gene sequence reading frames, constructing a successful pPIC9K-COL17-HR recombinant expression vector, and sequencing by a division of biological engineering (Shanghai) Co.
(2) The correct pPIC9K-COL17-HR expression vector was transformed into E.coli BL21 (DE 3) (from Shanghai) and the single-spotted clones were screened with ampicillin sodium-LB plates, cultured overnight in 5ml LB medium, transferred at 1:100, cultured in shake flasks at 37℃to an OD600 of 0.5, IPTG stock at 1:5000, cultured at 20℃for 8 hours, collected by centrifugation and stored at-20℃or immediately subjected to further purification.
(3) Washing the mixed bacterial precipitate with PBS buffer solution, re-suspending about 500ml bacterial precipitate with 20-40ml volume, decomposing bacteria with lysozyme and Triton X-100, ultrasonically decomposing bacteria under ice-water mixture environment for 60min, centrifuging at 12000rpm/min for 20min, and collecting supernatant. The solution contains a large amount of target recombinant XVII-type humanized collagen.
(4) Ammonium sulfate precipitation, fractional salting-out and preliminary purification are selected, and the optimal saturation of ammonium sulfate salting-out is 20% after searching for the saturation of ammonium sulfate. Washing a nickel ion affinity column (Ni-NTA His-Bind Resin) column material by using a PBS buffer solution, mixing the column material and the desalted and purified solution, carrying out co-culture, carrying out light shaking on the mixture at room temperature or on ice for 30min, then loading the column, washing the hybrid protein by using a PBS solution containing 15mM imidazole, leaving pure XVII type collagen on the column, adding a proper amount of Prescission Protease protease with His tag on the column, carrying out light shaking on the column at room temperature or on ice for 2 h, releasing the XVII type collagen from the column, eluting the column by using the PBS solution, and obtaining the recombinant XVII type humanized collagen with high purity SEQ ID NO:1, carrying out freeze-drying for later use, and using SDS-PAGE electrophoresis to verify the molecular weight of the protein.
SDS-PAGE electrophoresis of the supernatant from step (3) and the purified recombinant XVII-type humanized collagen solution is carried out, and the result is shown in FIG. 1, wherein lane 1 is the supernatant sample from step (3), and lane 2 is the purified recombinant XVII-type humanized collagen sample. The theoretical molecular weight of the recombinant XVII type humanized collagen COL17-HR is 50.55KD, the size of an electrophoresis pattern band 2 is about 50KD, and the size is consistent with the theoretical molecular weight, so that the recombinant XVII type humanized collagen COL17-HR is proved to be successfully expressed, and the purified protein presents a single band and has no degradation.
EXAMPLE 3 evaluation of cytotoxicity of recombinant XVII-type humanized collagen
HeLa cells grown to 80% of the bottom area of the culture flask were digested with 0.25% pancreatin, and a cell suspension having a cell density of 1X 10 5 cells/mL was prepared from the whole culture broth. 100. Mu.L of the cell suspension was inoculated into a 96-well culture plate and cultured in an incubator at 37℃under a saturated humidity of 5% CO 2. After 24h of cell culture, the complete culture broth was aspirated. Recombinant XVII-type humanized collagen solution (3 multiple wells are arranged for each concentration) with 10 mug/ml and 100 mug/ml concentration diluted by high-sugar DMEM culture medium is added into an experimental group, a control group is cells cultured by the DMEM culture medium, a blank group is the DMEM culture medium without cells, and the culture is continued for 24 hours in an incubator with saturated humidity of 5% CO 2 at 37 ℃. To each group, 10. Mu.L of CCK-8 reagent was added, and the mixture was incubated in a cell culture incubator for 2 hours, and the absorbance (OD value) of each well was measured at a wavelength of 450nm using an ELISA. Cell viability was calculated from the absorbance mean of each group according to the following formula:
；
In this example, the control group was a commercially available recombinant XVII type collagen sample solution added to the cell culture broth at 100. Mu.g/ml, 3 replicates per group.
The experimental results are shown in figure 4, and the cell viability of each experimental group is over 100%, which indicates that the recombinant XVII type humanized collagen prepared by the invention has no cytotoxicity and good safety.
EXAMPLE 4 evaluation of recombinant XVII-type humanized collagen cell proliferation-promoting Activity
BALB/c 3T3 cells (mouse embryo fibroblasts) with good logarithmic growth phase state (purchased from Shanghai cell bank of China academy of sciences) are selected, inoculated into a 96-well cell culture plate, digested by 0.25% trypsin, centrifuged, resuspended in DMEM medium containing 10% fetal bovine serum and counted; BALB/c 3T3 cells were seeded in 96-well plates at a cell number of approximately 5X 10 3 per well and a blank without cells was placed and incubated overnight at 37℃in a 5% CO 2 environment. The recombinant XVII-type humanized collagen prepared in example 2 was added at final concentrations of 10. Mu.g/ml and 100. Mu.g/ml, respectively, and each group was incubated for 48h at 37℃in a 5% CO 2 environment. Adding 5g/L MTT solution 4h before the culture is finished, and stopping the culture after continuing the culture for 4 h; the liquid in the wells was removed, washed 1 time with PBS, dissolved in DMSO to crystallize, and absorbance values were measured at 570 nm.
Cell proliferation rate= (measured well absorbance value-blank absorbance value)/(cell control absorbance value-blank absorbance value) ×100%.
In this example, the control group was a commercially available recombinant XVII type collagen sample solution added to the cell culture broth at 100. Mu.g/ml, 3 replicates per group.
As shown in FIG. 5, the experimental results show that the proliferation promoting rate of the control group and the recombinant XVII type humanized collagen of the application on BALB/c 3T3 cells is 67.4% and 124.6% respectively at the concentration of 100 mug/ml, and both are better than 35.8% of the recombinant XVII type humanized collagen of the application at the concentration of 10 mug/ml, which shows that the recombinant XVII type humanized collagen prepared by the application can obviously promote proliferation of fibroblasts, and the effect is better than that of the commercially available recombinant XVII type collagen.
EXAMPLE 5 evaluation of recombinant XVII-type humanized collagen cell adhesion Activity
The recombinant XVII-type humanized collagen prepared in example 2 was dissolved in PBS and added at final concentrations of 10. Mu.g/ml and 100. Mu.g/ml, respectively, and 50. Mu.L of the recombinant XVII-type humanized collagen solution was added to each well of a 96-well cell culture plate, and the mixture was left at 37℃for 2 hours, and PBS was added to the control well. BALB/3T3 cells were digested with pancreatin, counted, 5X 10 4 cells were added per well and incubated for 2h at 37℃in a 5% CO 2 incubator. Washing with PBS for 3 times, washing off non-adhered cells, and adding 200 mu LDMEM of culture medium; mu.L of CCK-8 reagent was added to each well, and the mixture was taken out after incubation in a 5% CO 2 cell incubator at 37℃for 2 hours. The absorbance values of the 96-well plate at 450nm and 630nm are read by an enzyme-labeled instrument, the absorbance is measured at 450nm by taking 630nm as a reference wavelength, and the measurement result is recorded.
Cell adhesion promotion rate= (experimental group 450nm absorbance-negative control group 450nm absorbance)/negative control group 450nm absorbance x 100%.
In this example, the control group was a commercially available recombinant XVII type collagen sample solution added to the cell culture broth at 100. Mu.g/ml, 3 replicates per group.
The experimental results are shown in FIG. 6, and the results show that the cell adhesion promoting rates of the control group and the recombinant XVII type humanized collagen of the application to BALB/c 3T3 cells are respectively 62.7% and 84.6% under the condition of 100 mug/ml, and both are better than 35.3% of the recombinant XVII type humanized collagen of the application with the concentration of 10 mug/ml, which shows that the recombinant XVII type humanized collagen prepared by the application has better cell adhesion promoting activity and better effect than the commercially available recombinant XVII type collagen.
Based on the above examples, the recombinant XVII type humanized collagen expressed by the present invention has uniform molecular weight, is non-toxic, has better cell adhesion and proliferation properties than the commercially available XVII type collagen, and can be applied to various fields such as pharmaceutical compositions, medical devices, tissue engineering products or cosmetics, etc.
The foregoing has outlined the basic principles, features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A recombinant XVII type humanized collagen is characterized in that the amino acid sequence of the collagen is shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the recombinant XVII-type humanized collagen of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No. 2.
4. A recombinant expression vector comprising the nucleic acid molecule of any one of claims 2-3.
5. A host cell comprising the recombinant expression vector of claim 4 or expressing the collagen of claim 1.
6. The host cell of claim 5, wherein the host cell is E.coli BL21 (DE 3).
7. The method for preparing recombinant XVII-type humanized collagen according to claim 1, comprising the steps of: (1) Culturing the host cell of claim 5 in a medium; (2) Isolating the humanized collagen of type XVII according to claim 1 from a host cell.
8. A composition comprising the recombinant XVII-type humanized collagen according to claim 1.
9. An article comprising the composition of claim 8, wherein the article is a pharmaceutical composition, a medical device, or a cosmetic.
10. Use of the composition of claim 8 for the preparation of a pharmaceutical composition, a medical device or a cosmetic.
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