Recombinant humanized XVII type collagen as well as expression vector and genetically engineered bacterium thereof

Abstract

The invention discloses a recombinant humanized XVII type collagen as well as an expression vector and a genetically engineered bacterium thereof. Four functional fragments with high activity are obtained through screening, one of the fragments is optimized and then combined according to a certain sequence to obtain recombinant humanized XVII type collagen JYC1703, or one of the fragments is optimized and then combined according to a certain sequence, and GPP sequences are fused at an amino terminal and a carboxyl terminal respectively to obtain recombinant humanized XVII type collagen JYC1701 and JYC1702, and the recombinant humanized XVII type collagen JYC1701 and JYC1702 are combined according to a certain sequence to obtain the recombinant humanized XVII type collagen JYC1701 and JYC1702. After codon optimization, amplifying and cloning into an expression vector, transforming into pichia pastoris competent cells after enzyme digestion linearization, and screening high-copy recombinants through G418 resistance gradient, so as to obtain pichia pastoris genetically engineered bacteria for expressing recombinant humanized XVII type collagen. The recombinant humanized XVII type collagen JYC1701, JYC1702 and JYC1703 disclosed by the invention show excellent functions of promoting cell adhesion, cell proliferation and cell migration, and are suitable for preparing products such as skin care products or medical instruments and the like.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a recombinant humanized type XVII collagen and an expression vector and genetic engineering bacteria thereof. 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 many types of collagen, type XVII collagen (COL17) has attracted much attention due to its key role in maintaining the adhesion between epidermal basal keratinocytes and the underlying dermis. This adhesion is essential for the integrity of the skin and its appendages, and defects in the function of type XVII collagen are associated with a range of severe blistering skin diseases, such as epidermolysis bullosa (EB) and blistering pemphigus (BP).

[0003] Type XVII collagen is a transmembrane protein that contains 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, including the difficulty 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 producing human-like collagen 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 their sequence and biological functions. Summary of the invention

[0005] The present invention obtains four highly active functional fragments, namely F1, F2, F3 and F4, by screening the functional domains binding to cell integrins in human type XVII collagen, and obtains recombinant humanized type XVII collagens JYC1703, JYC1702 and JYC1701 by optimizing F2 and combining the four functional fragments, or further fusing the GPP sequences at the amino termini and carboxyl termini of the combined fragments, amplifying and cloning them into a pPIC9K vector after codon optimization, and transforming them into Pichia pastoris cells after enzyme linearization, and screening high-copy recombinants through a G418 resistance gradient, thereby obtaining high-copy Pichia pastoris genetic engineering 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] The four functional fragments are F1, F2, F3 and F4, whose sequences are 100% identical to 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] The recombinant humanized type XVII collagen is JYC1701, JYC1702 or JYC1703; the JYC1703 is composed of the above-mentioned F1, the optimized F2, F3 and F4 four functional fragments combined in a certain order after optimizing the protease cleavage site and the non-GXY amino acid residues in the functional fragment F2, and its amino acid sequence is shown in SEQ ID No.5; the JYC1701 is composed of 7 repeats and 6 repeats of GPP sequences fused to the amino terminus and the carboxyl terminus of JYC1703 respectively, and its amino acid sequence is shown in SEQ ID No.6; the JYC1702 is composed of the above-mentioned F1, the optimized F2, F3 and F4 four functional fragments combined in a certain order after optimizing only the non-GXY amino acid residues in the functional fragment F2 to obtain a fragment carrying multiple integrin binding active sites, and 7 repeats and 6 repeats of GPP sequences are fused to the amino terminus and the carboxyl terminus of the combined fragment respectively, and its amino acid sequence is shown in SEQ ID No.7.

[0009] The nucleotide sequence of the gene encoding the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 is shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10.

[0010] A recombinant expression vector, which is a recombinant expression vector containing the gene encoding the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703. In a specific embodiment of the present invention, the recombinant expression vector pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703 containing the gene encoding the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 is taken as an example.

[0011] The method for constructing the above-mentioned recombinant expression vector comprises the following specific steps:

[0012] The gene sequence encoding the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10 was amplified by high-fidelity PCR, and the target fragment after gel recovery and purification was seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector, and enriched after colony PCR and sequencing verification to obtain the recombinant expression vector pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703.

[0013] The genetically engineered Pichia pastoris bacteria that produce the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 contain the above-mentioned recombinant expression vector. In a specific embodiment of the present invention, the recombinant expression vector pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703 is taken as an example.

[0014] The construction method of the above-mentioned Pichia pastoris genetically engineered bacteria comprises the following steps:

[0015] The above recombinant expression vector was linearized by enzyme digestion and transformed into Pichia pastoris GS115 competent cells, positive transformants were selected, and high-copy recombinants were screened by G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703.

[0016] In a specific embodiment of the present invention, the construction method of the above-mentioned Pichia pastoris genetically engineered bacteria comprises the following steps:

[0017] The above-mentioned recombinant expression vector pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703 is linearized by restriction digestion and transformed into Pichia pastoris GS115 competent cells, positive transformants are selected, and high-copy recombinants are screened by G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce 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 comprises the following steps:

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

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

[0021] A product comprising one or more of the above-mentioned recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703, wherein the product 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] The present invention obtains four highly active functional fragments (F1, F2, F3, and F4) by screening the functional domains binding to cell integrins in human type XVII collagen, and after optimizing the protease cleavage site and non-GXY amino acid residues in F2, the four 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 optimizing the protease cleavage site and non-GXY amino acid residues, the four functional fragments are combined in a certain order again and GPP sequences are fused to the amino terminal and the carboxyl terminal, respectively, to obtain recombinant humanized type XVII collagen JYC1702 and JYC1701. The invention performs codon optimization on the coding gene sequences of recombinant humanized type XVII collagens JYC1701, JYC1702 and JYC1703 according to the codon preference of Pichia pastoris, constructs a recombinant expression vector, transfers the vector into a Pichia pastoris strain, screens high-copy recombinants through a G418 resistance gradient, obtains a high-copy Pichia pastoris genetic engineering bacterium capable of producing the recombinant humanized type XVII collagens JYC1701, JYC1702 and JYC1703, and realizes the large-scale production of the recombinant humanized type XVII collagens JYC1701, JYC1702 and JYC1703. Cell experiments have found that recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 all have the ability to significantly promote the adhesion, proliferation and migration of human fibroblasts, epidermal cells and hair follicle stem cells. They can be used to promote the repair of skin and other tissues, enhance the resistance of the skin, and are suitable for the preparation of skin care or medical devices and other products. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 The electrophoresis diagram of plasmids expressing recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703, wherein (a) Lane 1: pPIC9K empty plasmid linearized fragment, Lane 2: pPIC9K+recombinant humanized type XVII collagen JYC1701 plasmid linearized fragment; (b) Lane 1: pPIC9K empty plasmid linearized fragment, Lane 2: pPIC9K+recombinant humanized type XVII collagen JYC1702 plasmid linearized fragment; (c) Lane 1: pPIC9K empty plasmid linearized fragment, Lane 2: pPIC9K+recombinant humanized type XVII collagen JYC1703 plasmid linearized fragment.

[0026] Figure 3 The present invention is an SDS-PAGE diagram of the supernatant of the culture medium of Pichia pastoris genetically engineered bacteria (methanol induced for 72 hours) for producing recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703, wherein (a) Lanes 1 to 6: SDS-PAGE of the supernatant of the high copy strain JYC1701, Lane 7: the fermentation supernatant of the blank control strain; (b) Lanes 1 to 6: SDS-PAGE of the supernatant of the high copy strain JYC1702, Lane 7: the fermentation supernatant of the blank control strain; (c) Lanes 1 to 8: SDS-PAGE of the supernatant of the high copy strain JYC1703, Lane 9: the fermentation supernatant of the blank control strain.

[0027] Figure 4 This is the total ion current of recombinant humanized type XVII collagen JYC1701.

[0028] Figure 5 This is a graph showing the molecular weight determination results of the recombinant humanized type XVII collagen JYC1701.

[0029] Figure 6 This figure shows 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 This figure shows 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 This figure shows 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 DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below through the preferred embodiments of the present invention, but the following embodiments do not limit the protection scope of the present invention.

[0033] In the embodiments of the present invention, conventional molecular biological experimental methods are used without further explanation. The processes such as PCR, enzyme cutting, seamless cloning, codon optimization, etc. involved in the embodiments are understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore 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) Using bioinformatics and protein structure prediction database tools (NCBI, AlphaFold), functional domains that bind to cell integrins were screened from human type XVII collagen, and four highly active functional fragments were obtained, namely F1 (amino acid sequence as shown in SEQ ID No.1), F2 (amino acid sequence as shown in SEQ ID No.2), F3 (amino acid sequence as shown in SEQ ID No.3) and F4 (amino acid sequence as shown in SEQ ID No.4). After optimizing the protease cleavage site and non-GXY amino acid residues in the functional fragment F2, the above-mentioned four functional fragments F1, optimized F2, F3 and F4 were combined in a certain order to obtain a recombinant humanized type XVII collagen JYC1703 carrying multiple integrin binding active sites, whose amino acid sequence is shown in SEQ ID No.5. Furthermore, 7 and 6 GPP sequences were fused to the amino and carboxyl ends of the recombinant humanized type XVII collagen JYC1703, respectively, to obtain the recombinant humanized type XVII collagen JYC1701, whose amino acid sequence is shown in SEQ ID No. 6. In addition, after optimizing only the non-GXY amino acid residues in the functional fragment F2, the above-mentioned 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 and 6 GPP sequences were fused to the amino and carboxyl ends of the combined fragments, respectively, to obtain the recombinant humanized type XVII collagen JYC1702, whose amino acid sequence is shown in SEQ ID No. 7.

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

[0037] (3) The nucleotide sequence in step (2) was entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis, and the recombinant humanized type XVII collagen JYC1701 gene sequence shown in SEQ ID No. 8 was amplified using primer P1 (nucleotide sequence as shown in SEQ ID No. 11) and primer P2 (nucleotide sequence as shown in SEQ ID No. 12); the recombinant humanized type XVII collagen JYC1702 gene sequence shown in SEQ ID No. 9 was amplified using primer P3 (nucleotide sequence as shown in SEQ ID No. 13) and primer P4 (nucleotide sequence as shown in SEQ ID No. 14); the recombinant humanized type XVII collagen JYC1703 gene sequence shown in SEQ ID No. 10 was amplified using primer P5 (nucleotide sequence as shown in SEQ ID No. 15) and primer P6 (nucleotide sequence as shown in SEQ ID No. 16). After high-fidelity PCR amplification products were purified by nucleic acid gel, they were seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid using the Gibson assembly technology. After verification by colony PCR and sequencing, the recombinant plasmids pPIC9K-JYC1701, pPIC9K-JYC1702, and pPIC9K-JYC1703 were enriched respectively. The structures of the recombinant plasmids pPIC9K-JYC1701, pPIC9K-JYC1702, and pPIC9K-JYC1703 are shown in the figure. Figure 1 As shown, the plasmid electrophoresis diagram is as follows Figure 2 Then, the recombinant plasmids pPIC9K-JYC1701, pPIC9K-JYC1702 and pPIC9K-JYC1703 were digested with SalI restriction endonuclease (purchased from NEB, the specific operation was carried out according to the kit instructions) at 37°C overnight, and then the linearized recombinant plasmids were purified using a gel recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and then electrotransformed into Pichia pastoris GS115 competent cells, and the electrotransformed cells were activated and cultured with fresh YPD medium for 2-3 hours, and then spread into YNB selection medium and placed in a 30°C incubator for inverted culture for 2-3 days to grow transformants. First, the single clone of the transformant was verified by colony PCR, and the correctly verified transformants were spread on screening plates containing 1g / L, 2g / L, 3g / L and 4g / L G418, respectively. After 2-4 days of cultivation, the transformants that grew well on the high-concentration G418 plates were picked out for purification and re-screening verification. The genome of the re-screened transformants was extracted and verified to be correct, and high-copy Pichia pastoris genetically engineered strains expressing recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 were obtained, and the seeds were preserved in glycerol.

[0038] (4) The high-copy Pichia genetically engineered strain obtained in step (3) was inoculated into 30 mL of BMGY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10× YNB 100 ml, 10× potassium phosphate buffer pH 6.0 100 ml, 10× glycerol 100 ml, and the balance was water), cultured for 16-20 hours, and then the initial OD was set at 0. 600 The inoculum size was 1 and inoculated into 30 mL BMMY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10×YNB 100 ml, 10× potassium phosphate buffer pH 6.0 100 ml, and the balance was water) for cultivation. 300 μL of filtered and sterilized methanol was added every 24 hours. After 72 hours, 2 mL of sample was centrifuged to collect the supernatant. 500 μL of supernatant was concentrated in an ultrafiltration tube with a size of 10 kDa to obtain about 50 μL of concentrated solution. 24 μL of concentrated solution and 6 μL of 5×loading buffer were mixed evenly and placed in boiling water for 10-15 min. After centrifugation for 15 min, 10-15 μL of supernatant was taken and loaded into 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 ,from Figure 3 It can be seen that a high-copy Pichia pastoris genetically engineered strain that can successfully express recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 was obtained, and it can be seen from the figure that the protein yield and stability of JYC1701 are significantly better than those of JYC1702 and JYC1703.

[0039] The target protein bands at the corresponding cutting positions were recovered and digested with trypsin, and the peptides after digestion were detected by LC-MS, and the detected peptides were compared with the library.

[0040] Take JYC1701 as an example. Figure 4 This is the total ion current chromatogram of the peptides of pure JYC1701 after trypsin digestion. It can be seen from the figure that the peptides of JYC1701 after 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 the target band are all derived from the collagen designed in the present invention, and JYC1701 is successfully and efficiently secreted outside the cell in Pichia pastoris. Figure 5 This is the result of mass spectrometry determination of 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 activity of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703

[0042] The MTT method was used to detect the ability of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 to promote cell proliferation using human epidermal cells, human fibroblasts and human hair follicle stem cells as experimental cells. Figure 6 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 1ppm, 10ppm, 100ppm, and 1000ppm of recombinant humanized type XVII collagen JYC1701, JYC1702, and JYC1703 all exhibited good cell proliferation promoting activity, and the promoting effect increased with the increase of 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 has stronger cell proliferation promoting activity.

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

[0044] The cell scratch method was used to measure cell migration. Human epidermal cells, human fibroblasts and human 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 migration. Figure 7 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 1ppm, 10ppm, 100ppm and 1000ppm of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 all show good cell migration promotion activity, and the promotion effect increases with the increase of 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 has stronger cell migration promotion activity.

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

[0046] The centrifugation method was used to detect the ability of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 to promote cell adhesion using human epidermal cells, human fibroblasts and hair follicle stem cells as experimental cells. Figure 8 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 10ppm, 100ppm and 1000ppm of recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 all show good cell adhesion promoting activity, and the promoting effect increases with the increase of 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 has stronger cell adhesion promoting activity.

Claims

1. Four functional fragments, characterized in that: They are F1, F2, F3 and F4 respectively, and 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.

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

7.

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

10.

4. A recombinant expression vector, characterized in that: A recombinant expression vector containing the gene encoding the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703 containing the gene encoding the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 according to claim 3 is constructed by the following steps: The gene sequence encoding the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10 was amplified by high-fidelity PCR, and the target fragment after gel recovery and purification was seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector, and enriched after colony PCR and sequencing verification to obtain the recombinant expression vector pPIC9K-JYC1701, pPIC9K-JYC1702 or pPIC9K-JYC1703.

6. The genetically engineered Pichia pastoris for producing the recombinant humanized type XVII collagen according to claim 2, characterized in that: Contains the recombinant expression vector according to claim 4 or 5.

7. The method for constructing a genetically engineered Pichia pastoris according to claim 6, characterized in that: The specific steps are as follows: The recombinant expression vector according to claim 4 or 5 is linearized and then transformed into Pichia pastoris GS115 competent cells, positive transformants are selected, and high-copy recombinants are screened through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703.

8. The method for preparing recombinant humanized type XVII collagen according to claim 2, characterized in that: Here are the steps: The genetically engineered Pichia pastoris producing the recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703 according to claim 6 is inoculated into a BMMY induction medium, induced to express with methanol, and the fermentation supernatant is purified to obtain high-purity recombinant humanized type XVII collagen JYC1701, JYC1702 or JYC1703.

9. A composition or product, characterized in that The invention comprises at least one of the recombinant humanized type XVII collagen JYC1701, JYC1702 and JYC1703 according to claim 2.

10. The composition or product according to claim 9, characterized in that The composition is a skin care product or a pharmaceutical composition, and the product is a medical device, a biomaterial or a tissue engineering product.

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