Recombinant humanized type XVII collagen, its expression vector and genetically engineered bacteria

Abstract

The present invention discloses a recombinant humanized type XVII collagen, its expression vector and genetically engineered bacteria. In the present invention, 4 functional fragments with high activity are obtained through screening. After optimizing one of the fragments and combining them in a certain order, recombinant humanized type XVII collagen JYC1703 is obtained. Or after optimizing one of the fragments, combining them in a certain order and respectively fusing GPP sequences at the amino terminus and carboxyl terminus, recombinant humanized type XVII collagens JYC1701 and JYC1702 are obtained. After codon optimization, they are amplified and cloned into an expression vector. After enzymatic digestion and linearization, they are transformed into Pichia pastoris competent cells. High-copy recombinants are screened through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria expressing recombinant humanized type XVII collagen. The recombinant humanized type XVII collagens JYC1701, JYC1702 and JYC1703 of the present invention exhibit excellent functions of promoting cell adhesion, cell proliferation and migration, and are suitable for preparing products such as skin care or medical devices.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and relates to a recombinant humanized type XVII collagen, its expression vector and genetically engineered bacteria. Background Art

[0002] Collagen is the main structural protein in the extracellular matrix (ECM), providing strength and elasticity to connective tissues such as skin, tendons, and cartilage. Among the numerous types of collagen, type XVII collagen (COL17) has attracted much attention because it plays a key role in maintaining the adhesion between epidermal basal keratinocytes and the underlying dermis. This adhesion is crucial for the integrity of the skin and its appendages, and functional defects of type XVII collagen are associated with a series of severe skin blistering diseases, such as epidermolysis bullosa (EB) and bullous pemphigus (BP).

[0003] Type XVII collagen is a transmembrane protein, containing an extracellular triple-helical domain and an intracellular domain anchored to the cell membrane. In particular, the extracellular triple-helical domain provides the necessary structural stability for its function in cell-matrix adhesion. However, despite the important biological functions of type XVII collagen, the production of its recombinant protein still faces challenges, such as difficulties in achieving high expression levels, correct folding, and functional activity.

[0004] Traditional recombinant protein expression systems, such as the Escherichia coli prokaryotic expression system, often have difficulty in producing human-like collagens with necessary post-translational modifications, correct folding, and structural stability. Therefore, improved methods are urgently needed to design and produce recombinant proteins that can accurately mimic natural human type XVII collagen and ensure the accuracy of its sequence and biological functions. Summary of the Invention

[0005] In the present invention, by screening the functional domains of human type XVII collagen that bind to cell integrins, 4 highly active functional fragments, namely F1, F2, F3, and F4, were obtained. By optimizing F2, combining the 4 functional fragments, or further fusing GPP sequences to the amino terminus and carboxyl terminus of the combined fragments respectively, recombinant humanized type XVII collagens JYC1703, JYC1702, and JYC1701 were obtained. After codon optimization, they were amplified and cloned into the pPIC9K vector, linearized by enzyme digestion, and then transformed into Pichia pastoris cells. High-copy recombinants were screened by G418 resistance gradient, thereby obtaining high-copy Pichia pastoris genetically engineered strains capable of expressing recombinant humanized type XVII collagens JYC1701, JYC1702, and JYC1703.

[0006] The technical solution of the present invention is as follows:

[0007] Four functional fragments, namely F1, F2, F3, and F4, have 100% identity with human type XVII collagen and have functional sites for binding to cell integrins. Their amino acid sequences are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4 respectively.

[0008] Recombinant humanized type XVII collagen is JYC1701, JYC1702, or JYC1703; JYC1703 is composed of the above four functional fragments F1, optimized F2, F3, and F4 in a certain order after optimizing the protease cleavage site and non-GXY amino acid residues in functional fragment F2, and its amino acid sequence is shown in SEQ ID No.5; JYC1701 is composed by fusing 7 repeats and 6 repeats of GPP sequences at the amino terminus and carboxyl terminus of JYC1703 respectively, and its amino acid sequence is shown in SEQ ID No.6; JYC1702 is composed by only optimizing the non-GXY amino acid residues in functional fragment F2, combining the above four functional fragments F1, optimized F2, F3, and F4 in a certain order to obtain a fragment carrying multiple integrin binding active sites, and fusing 7 repeats and 6 repeats of GPP sequences at the amino terminus and carboxyl terminus of the combined fragment respectively, and its amino acid sequence is shown in SEQ ID No.7.

[0009] The genes encoding the above recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703 have nucleotide sequences shown in SEQ ID No.8, SEQ ID No.9, or SEQ ID No.10 respectively.

[0010] A recombinant expression vector is a recombinant expression vector containing the genes encoding the above recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703. In the specific embodiments of the present invention, the recombinant expression vectors pPIC9K-JYC1701, pPIC9K-JYC1702, or pPIC9K-JYC1703 containing the genes encoding the above recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703 are taken as examples.

[0011] The construction method of the above recombinant expression vector is as follows:

[0012] Perform high-fidelity PCR amplification on the gene sequences encoding recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703 shown in SEQ ID No.8, SEQ ID No.9, or SEQ ID No.10. After gel recovery and purification of the target fragment, seamlessly clone it downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector. After enrichment through colony PCR and correct sequencing verification, obtain the recombinant expression vectors pPIC9K-JYC1701, pPIC9K-JYC1702, or pPIC9K-JYC1703.

[0013] Construct Pichia pastoris genetic engineering bacteria for producing the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703, which contain the above-mentioned recombinant expression vector. In the specific embodiments of the present invention, take the recombinant expression vectors pPIC9K-JYC1701, pPIC9K-JYC1702, or pPIC9K-JYC1703 as examples.

[0014] The construction method of the above-mentioned Pichia pastoris genetic engineering bacteria is as follows:

[0015] Linearize the above-mentioned recombinant expression vector by enzyme digestion and then transform it into Pichia pastoris GS115 competent cells. Select positive transformants and screen for high-copy recombinants through G418 resistance gradient to obtain Pichia pastoris genetic engineering bacteria for producing recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703.

[0016] In the specific embodiments of the present invention, the construction method of the above-mentioned Pichia pastoris genetic engineering bacteria is as follows:

[0017] Linearize the above-mentioned recombinant expression vectors pPIC9K-JYC1701, pPIC9K-JYC1702, or pPIC9K-JYC1703 by enzyme digestion and then transform them into Pichia pastoris GS115 competent cells. Select positive transformants and screen for high-copy recombinants through G418 resistance gradient to obtain Pichia pastoris genetic engineering bacteria for producing recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703.

[0018] The preparation method of the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702, or JYC1703 is as follows:

[0019] The Pichia pastoris genetic engineering bacteria for producing recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 are inoculated into the BMMY induction medium, induced and expressed by methanol, and the fermentation supernatant is purified to obtain high-purity recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703.

[0020] A composition comprising one or more of the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703, and the composition includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0021] An article comprising one or more of the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703, and the article includes but is not limited to medical devices, biomaterials, tissue engineering products, etc.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] In the present invention, by screening the functional domains that bind to cell integrin in human type XVII collagen, 4 highly active functional fragments (F1, F2, F3, F4) are obtained. After optimizing the protease cleavage site and non-GXY amino acid residues in F2, the 4 functional fragments are combined in a certain order to obtain recombinant humanized type XVII collagen JYC1703, or after optimizing the non-GXY amino acid residues in F2, or after optimizing the protease cleavage site and non-GXY amino acid residues, the 4 functional fragments are combined in a certain order and GPP sequences are fused at the amino terminus and carboxyl terminus respectively to obtain recombinant humanized type XVII collagen JYC1702 and JYC1701. In the present invention, the coding gene sequences of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 are codon-optimized according to the codon preference of Pichia pastoris, a recombinant expression vector is constructed, and it is transferred into Pichia pastoris strains. High-copy recombinants are screened by G418 resistance gradient to obtain high-copy Pichia pastoris genetic engineering bacteria capable of producing recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703, realizing the large-scale production of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703. Cell experiments found that recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 all have the effects of significantly promoting the adhesion, proliferation and migration of human fibroblasts, epidermal cells and hair follicle stem cells, can be used to promote the repair of tissues such as skin, enhance the resistance of the skin, and are suitable for preparing products such as skin care or medical devices. Description of the Drawings

[0024] Figure 1 Schematic diagram of plasmid structures for expressing recombinant humanized type XVII collagen JYC1701(a), JYC1702(b), and JYC1703(c).

[0025] Figure 2 Electrophoresis patterns of plasmids for expressing recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703. In panel (a), lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K + recombinant humanized type XVII collagen JYC1701 plasmid. In panel (b), lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K + recombinant humanized type XVII collagen JYC1702 plasmid. In panel (c), lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K + recombinant humanized type XVII collagen JYC1703 plasmid.

[0026] Figure 3 SDS-PAGE patterns of culture supernatants (induced with methanol for 72 hours) of Pichia pastoris genetic engineering bacteria for producing recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703. In panel (a), lanes 1 - 6: SDS-PAGE of supernatant of high-copy strain JYC1701; lane 7: fermentation supernatant of blank control strain. In panel (b), lanes 1 - 6: SDS-PAGE of supernatant of high-copy strain JYC1702; lane 7: fermentation supernatant of blank control strain. In panel (c), lanes 1 - 8: SDS-PAGE of supernatant of high-copy strain JYC1703; lane 9: fermentation supernatant of blank control strain.

[0027] Figure 4 Total ion current chromatogram of recombinant humanized type XVII collagen JYC1701.

[0028] Figure 5 Results graph of molecular weight determination of recombinant humanized type XVII collagen JYC1701.

[0029] Figure 6 Results graphs of the effects of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 on the proliferation of epidermal cells, fibroblasts, and hair follicle stem cells.

[0030] Figure 7 Results graphs of the effects of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 on the migration of epidermal cells, fibroblasts, and hair follicle stem cells.

[0031] Figure 8 Results of the effects of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 on the adhesion of epidermal cells, fibroblasts, and hair follicle stem cells Detailed implementation mode

[0032] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be elaborated in detail below through the preferred embodiments of the present invention. However, the following embodiments do not limit the protection scope of the present invention.

[0033] In the embodiments of the present invention, those not described in detail are completed by using conventional molecular biology experimental methods. Processes such as PCR, restriction enzyme digestion, seamless cloning, and codon optimization involved in the embodiments can be understood and easily implemented by those skilled in the art according to the product specifications or basic knowledge in the art, and thus will not be described in detail.

[0034] Example 1: Design and construction of high-copy Pichia pastoris genetic engineering bacteria of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703

[0035] (1) Screening for functional domains that bind to cell integrins from human type XVII collagen using bioinformatics and protein structure prediction database tools (NCBI, AlphaFold), four highly active functional fragments were obtained, namely F1 (amino acid sequence shown in SEQ ID No.1), F2 (amino acid sequence shown in SEQ ID No.2), F3 (amino acid sequence shown in SEQ ID No.3), and F4 (amino acid sequence shown in SEQ ID No.4). After optimizing the protease cleavage sites and non-GXY amino acid residues in functional fragment F2, the above four functional fragments, F1, optimized F2, F3, and F4, were combined in a certain order to obtain recombinant humanized type XVII collagen JYC1703 carrying multiple integrin-binding active sites, and its amino acid sequence is shown in SEQ ID No.5. Further, 7 repeats and 6 repeats of GPP sequences were fused to the amino terminus and carboxyl terminus of recombinant humanized type XVII collagen JYC1703 respectively to obtain recombinant humanized type XVII collagen JYC1701, and its amino acid sequence is shown in SEQ ID No.6. In addition, after only optimizing the non-GXY amino acid residues in functional fragment F2, the above four functional fragments, F1, optimized F2, F3, and F4, were combined in a certain order to obtain a fragment carrying multiple integrin-binding active sites, and 7 repeats and 6 repeats of GPP sequences were fused to the amino terminus and carboxyl terminus of the combined fragment respectively to obtain recombinant humanized type XVII collagen JYC1702, and its amino acid sequence is shown in SEQ ID No.7.

[0036] (2) The amino acid sequences of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 obtained in step (1) were codon-optimized according to the codon preference of Pichia pastoris to obtain the nucleotide sequences of the genes encoding recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 after optimization, which are shown in SEQ ID No.8, SEQ ID No.9, and SEQ ID No.10 respectively.

[0037] (3) Entrust the nucleotide sequence in step (2) to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis, and amplify the recombinant humanized type XVII collagen JYC1701 gene sequence shown in SEQ ID No. 8 with primer P1 (nucleotide sequence as shown in SEQ ID No. 11) and primer P2 (nucleotide sequence as shown in SEQ ID No. 12); amplify the recombinant humanized type XVII collagen JYC1702 gene sequence shown in SEQ ID No. 9 with primer P3 (nucleotide sequence as shown in SEQ ID No. 13) and primer P4 (nucleotide sequence as shown in SEQ ID No. 14); amplify the recombinant humanized type XVII collagen JYC1703 gene sequence shown in SEQ ID No. 10 with primer P5 (nucleotide sequence as shown in SEQ ID No. 15) and primer P6 (nucleotide sequence as shown in SEQ ID No. 16). After purifying the high-fidelity PCR amplification products by nucleic acid gel, use Gibson assembly technology to seamlessly clone them downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid. After verifying the correctness by colony PCR and sequencing, enrich the recombinant plasmids pPIC9K-JYC1701, pPIC9K-JYC1702, and pPIC9K-JYC1703 respectively. The structures of the recombinant plasmids pPIC9K-JYC1701, pPIC9K-JYC1702, and pPIC9K-JYC1703 are as Figure 1 shown, and the plasmid electrophoresis pattern is as Figure 2 shown. Then, use SalI restriction endonuclease (purchased from NEB, and the specific operation is carried out according to the kit instructions) to digest the recombinant plasmids pPIC9K-JYC1701, pPIC9K-JYC1702, and pPIC9K-JYC1703 overnight at 37 °C. After that, use a gel extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to purify the linearized recombinant plasmids and then electrotransform them into the competent cells of Pichia pastoris GS115. Activate the electrotransformed cells with fresh YPD medium for 2-3 hours, and then coat them on the YNB selection medium and place them in an incubator at 30 °C for inverted culture for 2-3 days until transformants grow. First, verify the transformant monoclonal by colony PCR. Coat the verified transformants on the screening plates containing 1 g / L, 2 g / L, 3 g / L, and 4 g / L G418 respectively. After culturing for 2-4 days, pick the transformants with better growth on the high-concentration G418 plates for purification and rescreening verification. After extracting the genomes of the transformants obtained by rescreening and verifying their correctness, obtain the high-copy Pichia pastoris genetic engineering strains expressing recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 respectively, and preserve them in glycerol.

[0038] (4) Inoculate the high-copy Pichia pastoris genetic engineering strain obtained in step (3) into 30 mL of BMGY medium (this medium consists of 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10× potassium phosphate buffer pH 6.0, 100 mL of 10× glycerol, and the balance is water), culture for 16 - 20 hours, and then inoculate it into 30 mL of BMMY medium (this medium consists of 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10× potassium phosphate buffer pH 6.0, and the balance is water) at an inoculum size with an initial OD 600 of 1 for culture. Supplement 300 μL of sterile-filtered methanol every 24 hours. After 72 hours, take 2 mL of the sample, centrifuge to collect the supernatant, pipette 500 μL of the supernatant into an ultrafiltration tube with a size of 10 kDa for concentration to obtain approximately 50 μL of concentrated solution. Mix 24 μL of the concentrated solution and 6 μL of 5× loading buffer evenly, place it in boiling water for 10 - 15 min, centrifuge for 15 min, and then pipette 10 - 15 μL of the supernatant for loading onto SDS-PAGE to complete the qualitative analysis of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703. The results of SDS-PAGE detection are shown in Figure 3 , and it can be seen from Figure 3 that a high-copy Pichia pastoris genetic engineering strain capable of successfully expressing recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 is obtained, and it can be seen from the figure that both the protein yield and stability of JYC1701 are significantly better than those of JYC1702 and JYC1703.

[0039] By recovering and cutting the target protein band at the corresponding position, digest it with trypsin, detect the peptides after digestion by liquid chromatography-mass spectrometry, and perform library comparison on the detected peptides.

[0040] Taking JYC1701 as an example, Figure 4 is the total ion chromatogram of the peptides after trypsin digestion of pure JYC1701. It can be seen from the figure that the peptides after JYC1701 digestion all belong to the region of the human type XVII collagen sequence selected during design, and the peptide coverage rate is 100%, proving that the peptides in this target band all originate from the collagen designed in the present invention, and JYC1701 is successfully and efficiently secreted extracellularly in Pichia pastoris. Figure 5 is the mass spectrometry result map for measuring the molecular weight. It can be seen from the figure that the actual molecular weight of JYC1701 is 17.8 kDa, which is consistent with the theoretical molecular weight.

[0041] Example 2: Detection of the cell proliferation-promoting activities of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703

[0042] Using the MTT method, the abilities of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 to promote cell proliferation were detected using human epidermal cells, human fibroblasts, and human hair follicle stem cells as experimental cells. The results are as Figure 6 shown. The blank group was a control experiment in which no components were added and only cell culture medium was used for culture. It can be seen that recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 at 1 ppm, 10 ppm, 100 ppm, and 1000 ppm all showed good cell proliferation-promoting activities, and the promoting effects were enhanced as the concentrations of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 increased. In addition, compared with recombinant humanized type XVII collagen JYC1702 and JYC1703, JYC1701 had stronger cell proliferation-promoting activity.

[0043] Example 3: Detection of the cell migration-promoting activities of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703

[0044] The cell scratch method was used to measure cell migration. The abilities of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 to promote cell migration were detected using human epidermal cells, human fibroblasts, and human hair follicle stem cells as experimental cells. The results are as Figure 7 shown. The blank group was a control experiment in which no components were added and only cell culture medium was used for culture. It can be seen that recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 at 1 ppm, 10 ppm, 100 ppm, and 1000 ppm all showed good cell migration-promoting activities, and the promoting effects were enhanced as the concentrations of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 increased. In addition, compared with recombinant humanized type XVII collagen JYC1702 and JYC1703, JYC1701 had stronger cell migration-promoting activity.

[0045] Example 4: Detection of the cell adhesion-promoting activities of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703

[0046] Using the centrifugation method, human epidermal cells, human fibroblasts, and hair follicle stem cells were used as experimental cells to detect the ability of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 to promote cell adhesion. The results are as Figure 8 shown. The blank group was a control experiment that only used cell culture medium without adding any components. It can be seen that recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 at 10 ppm, 100 ppm, and 1000 ppm all showed good cell adhesion-promoting activity, and the promoting effect increased with the increase in the concentration of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703. In addition, compared with recombinant humanized type XVII collagen JYC1702 and JYC1703, JYC1701 had stronger cell adhesion-promoting activity.

Claims

1. Recombinant humanized type XVII collagen, characterized in that, It is JYC1701, JYC1702 or JYC1703; the amino acid sequence of JYC1703 is shown as SEQ ID No.5, the amino acid sequence of JYC1701 is shown as SEQ ID No.6, and the amino acid sequence of JYC1702 is shown as SEQ ID No.

7.

2. The gene encoding the recombinant humanized type XVII collagen according to claim 1, characterized in that, The nucleotide sequence of the gene encoding recombinant humanized type XVII collagen JYC1701 is shown as SEQ ID No.8, the nucleotide sequence of the gene encoding recombinant humanized type XVII collagen JYC1702 is shown as SEQ ID No.9, and the nucleotide sequence of the gene encoding recombinant humanized type XVII collagen JYC1703 is shown as SEQ ID No.

10.

3. A recombinant expression vector, characterized in that, It is a recombinant expression vector containing the gene encoding recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 as described in claim 2.

4. The recombinant expression vector according to claim 3, wherein It is the recombinant expression vectors pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703 containing the gene encoding recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 as described in claim 2, and is constructed by the following steps: Perform high-fidelity PCR amplification on the gene sequences encoding recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 shown as SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10, and seamlessly clone the target fragment purified by gel recovery downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector, and enrich it after verification by colony PCR and sequencing to obtain the recombinant expression vectors pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703.

5. A Pichia pastoris genetic engineering bacterium for producing the recombinant humanized type XVII collagen described in claim 1, characterized in that, It contains the recombinant expression vector as described in claim 3 or 4.

6. The construction method of the Pichia pastoris genetic engineering bacterium according to claim 5, characterized in that, The specific steps are as follows: Linearize the recombinant expression vector as described in claim 3 or 4 by enzyme digestion and transform it into the competent cells of Pichia pastoris GS115, select positive transformants, and screen for high-copy recombinants through G418 resistance gradient to obtain the Pichia pastoris genetic engineering bacteria for producing recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703.

7. The preparation method of the recombinant humanized type XVII collagen according to claim 1, characterized in that, The steps are as follows: Inoculate the Pichia pastoris genetic engineering bacteria for producing recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 as described in claim 5 into the BMMY induction medium, induce expression with methanol, and purify the fermentation supernatant to obtain high-purity recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703.

8. A composition, characterized in that, It contains more than one of the recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 as described in claim 1.

9. The composition according to claim 8, wherein The composition is a skin care product or a pharmaceutical composition.

10. An article, characterized in that, Comprising one or more of the recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 as described in claim 1.

11. The article according to claim 10, characterized in that, The article is a medical device.

12. The article according to claim 10, characterized in that, The article is a biomaterial.

13. The article according to claim 10, characterized in that, The article is a tissue engineering product.

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