Debridge-(X VII) type collagen fusion protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to desmosome-XVII type collagen fusion protein as well as a preparation method and application thereof. The invention provides a recombinant XVII type collagen fusion protein which comprises at least one XVII type collagen functional domain fragment and at least one functional domain fragment of desmosome core glycoprotein, the recombinant human X VII type collagen fusion protein provided by the invention has better cell adhesion activity and cell proliferation promoting activity. The recombinant human XVII type collagen fusion desmosis protein can significantly improve the adhesion between cells in a cell culture environment, and compared with commercial human XVII type collagen, the recombinant human XVII type collagen fusion desmosis protein can improve the effect by several times or more. In the aspect of promoting cell proliferation, the cell division cycle can be effectively shortened, so that the number of cells is rapidly increased within the same time, and the XVII type collagen is remarkably superior to commercial human XVII type collagen, and has wider application potential in the fields of skin care products, cosmetics, medicines, biomedical materials and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a desmosome-type XVII collagen fusion protein, a preparation method and an application thereof. Background Art

[0002] Type XVII collagen, also known as type XVII collagen, is a member of the collagen family and a transmembrane structural protein. This protein is primarily distributed in the basement membranes of various tissues, including the skin, kidneys, lungs, and cartilage. Type XVII collagen plays a vital role in cell attachment and signal transduction, particularly in the connection between epithelial and mesenchymal cells, where it plays a central role. The protein is composed of three different α (alpha) chains, which are tightly linked by a triple helical structure. Type XVII collagen has a significant impact on numerous physiological and pathological processes. It participates in cell differentiation, migration, and proliferation, and is crucial for maintaining the normal structure and function of tissues.

[0003] Unlike other types of collagen, type XVII collagen, also known as anchoring fibers, constitutes the primary anchoring fiber component of the epidermal-dermal basement membrane zone. Type XVII collagen (encoded by the COL17A1 gene) is a 1,497-amino acid transmembrane protein that forms a structural component of hemidesmosomes and plays a key role in the interaction between epithelial cells and the basement membrane. It regulates epithelial cell adhesion, separation, development, and differentiation, and has a crucial influence on keratinocyte differentiation and regeneration.

[0004] Currently, relevant research reports have been published, demonstrating that using genetic engineering techniques to construct a biological system for recombinant human type XVII collagen can produce recombinant type XVII collagen with biological properties, which can be used in skincare products and skin repair medical materials. However, the cell adhesion and cell proliferation promoting effects of currently commercialized recombinant human type XVII collagen remain limited, and its functions are relatively limited. Summary of the Invention

[0005] Therefore, the technical problem addressed by the present invention is to provide a desmosome-type XVII collagen fusion protein, a preparation method, and its use. This invention rationally designs a desmosome-type XVII collagen fusion protein with high biological properties and successfully achieves efficient expression in a heterologous host. This achievement will greatly promote the application of type XVII collagen in industrial production.

[0006] To this end, the present invention provides the following technical solutions:

[0007] An embodiment of the present invention provides a recombinant type XVII collagen fusion protein, comprising: at least one type XVII collagen functional region fragment, and at least one desmosome core glycoprotein functional region fragment.

[0008] Desmosomal DSG protein, as a member of the desmosomal cadherin family, is a crucial intercellular junction protein in the skin and other epithelial tissues. Like other members of the family, it undertakes the responsibilities of intercellular adhesion and signal transmission to ensure the integrity of tissue structure and the normal functioning of functions. As a signaling protein, desmosomal cadherin participates in the proliferation, differentiation and morphogenesis of cells. Among them, desmoglein (Dsg) and desmocollin (Dsc) are the two main transmembrane glycoproteins in desmosomes. They are both N-glycosylated type I transmembrane proteins (i.e., the N-terminus is outside the cell and the C-terminus is inside the cell), and belong to the Ca 2+ The cadherin family of cell-dependent adhesion factors. Studies have shown that DSG1 plays a role in the immune response of the skin and can regulate the activation and infiltration of immune cells in the skin. This is of great significance for controlling skin inflammation and immune-related diseases. DSG1 is a member of the cadherin family and is mainly found in the desmosomes of the skin. Its role in the field of skin care is mainly reflected in maintaining the skin barrier function and participating in immune regulation. Among them, DSG1 (GenBank: AAC83817.1) is composed of 1049 amino acids, has a large molecular weight, and contains 4 domains and 5 desmoglein repeats. Type XVII collagen is an important basement membrane component that participates in cell attachment and signal transduction, and desmosome proteins are key proteins for cell-to-cell connection. Therefore, by combining the functional domains of desmosome-associated proteins with type XVII collagen, the fusion protein possesses the functions of both desmosome-associated proteins and type XVII collagen, which helps to enhance the cell-cell adhesion mediated by desmosome-associated proteins and promote wound healing. It may also help to enhance the adhesion between cells and the basement membrane, promote cell proliferation and differentiation, and thus achieve tissue repair and regeneration.

[0009] In some embodiments, the type VII collagen functional region fragment is selected from any one of the following amino acid sequences (1)-(2):

[0010] (1) having an amino acid sequence as shown in any one of SEQ ID NO. 1;

[0011] (2) An amino acid sequence having an identity of 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the amino acid sequence shown in any one of SEQ ID NO.1 described in (1).

[0012] In some embodiments, the functional region fragment of the desmosome core glycoprotein is selected from any one of the following amino acid sequences (1)-(2):

[0013] (1) having the amino acid sequence shown in SEQ ID NO. 2;

[0014] (2) An amino acid sequence having an identity of 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the amino acid sequence shown in SEQ ID NO. 2 described in (1).

[0015] In some embodiments, the recombinant type XVII collagen fusion protein further contains a type III collagen expression-promoting sequence;

[0016] Optionally, the type III collagen expression-promoting sequence is selected from any one of the following amino acid sequences (1)-(2):

[0017] (1) having the amino acid sequence shown in SEQ ID NO. 3;

[0018] (2) An amino acid sequence having an identity of 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the amino acid sequence shown in SEQ ID NO. 3 described in (1).

[0019] In some embodiments, the C-terminus of the XVII type collagen functional domain fragment is fused to the functional domain fragment of the desmosome core glycoprotein;

[0020] Optionally, the N-terminus of the type XVII collagen functional region fragment is fused with the type III collagen expression-promoting sequence;

[0021] Optionally, the functional domain fragment of type XVII collagen and the functional domain fragment of the desmosome core glycoprotein are connected by at least one linker; preferably, the number of linkers is 1-3;

[0022] Optionally, the type III collagen expression-promoting sequence and the type XVII collagen functional region fragment are connected by at least one linker; preferably, the number of linkers is 1-3;

[0023] Optionally, the amino acid sequence of the linker is GGGGS.

[0024] An embodiment of the present invention provides a biomaterial, including any one of the following:

[0025] 1) A nucleic acid molecule encoding the recombinant type XVII collagen fusion protein; optionally, the nucleic acid molecule is DNA or RNA;

[0026] 2) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line expressing the recombinant type XVII collagen fusion protein;

[0027] 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in 1);

[0028] 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in 2) or 3);

[0029] 5) A host cell containing the recombinant vector described in 2) or 3) or 4).

[0030] In some embodiments, the initial plasmid vector of the recombinant vector is a pPIC9K plasmid;

[0031] and / or, the host cell comprises yeast;

[0032] And / or, the method for constructing the recombinant vector comprises: inserting a nucleic acid molecule encoding the recombinant type XVII collagen fusion protein into a plasmid vector through an enzyme cleavage site to obtain the recombinant vector;

[0033] And / or, the method for constructing the host cell containing the recombinant vector comprises: linearizing the recombinant vector by enzyme digestion, then transferring the recombinant vector into competent host cells, culturing, and screening positive transformants.

[0034] An embodiment of the present invention provides a method for preparing the recombinant type XVII collagen fusion protein, characterized by comprising:

[0035] Synthesizing the coding gene for the recombinant type XVII collagen fusion protein;

[0036] constructing a recombinant vector containing the encoding gene;

[0037] The recombinant vector is transformed into host cells and fermented and cultured.

[0038] In some embodiments, the initial plasmid vector of the recombinant vector is a pPIC9K plasmid;

[0039] and / or, the host cell comprises yeast;

[0040] And / or, the fermentation culture conditions include any one of the following:

[0041] The inoculation rate is 8% to 10%;

[0042] Stirring speed 200-400 rpm;

[0043] Tank pressure 0.03~0.05MPa;

[0044] Control pH 5.0-6.0;

[0045] Temperature 25-30℃;

[0046] Dissolved oxygen is controlled at 25-35%;

[0047] And / or, after the fermentation reaches the set value, methanol is added for induction culture, the pH is adjusted to 5.0-6.0, the dissolved oxygen is controlled at 25-35%, and the induction is carried out for 48-72 hours, preferably 48 hours.

[0048] The embodiments of the present invention provide a use of the recombinant type XVII collagen fusion protein, the biomaterial, or the recombinant type XVII collagen fusion protein prepared by the preparation method, including any of the following:

[0049] (1) Promoting cell proliferation or preparing products that promote cell proliferation;

[0050] (2) promoting cell adhesion or preparing products that promote cell adhesion;

[0051] (3) promoting cell migration or preparing products that promote cell migration;

[0052] (4) Preparation of skin care products or cosmetics;

[0053] (5) Preparation of biomedical materials;

[0054] Optionally, the product includes skin care products, cosmetics, medicines or biomedical materials.

[0055] The technical solution of the present invention has the following advantages:

[0056] 1. The present invention provides a recombinant type XVII collagen fusion protein, comprising: at least one functional region fragment of type XVII collagen, and at least one functional region fragment of the core glycoprotein of the desmosome; the recombinant human type XVII collagen fusion protein provided by the present invention has better cell adhesion activity and cell proliferation promoting activity. Through a series of experiments, it was verified that the recombinant human type XVII collagen fusion desmosome protein can significantly improve the adhesion between cells in a cell culture environment, and the improvement effect can be several times higher than that of commercial human type XVII collagen. In terms of promoting cell proliferation, it can effectively shorten the cell division cycle, so that the cell number increases rapidly within the same time, which is significantly better than commercial human type XVII collagen. In summary, the recombinant human type XVII collagen fusion protein has a wider application potential in the fields of skin care products, cosmetics, drugs and biomedical materials.

[0057] 2. The present invention provides a recombinant type XVII collagen fusion protein, which also contains a type III collagen expression-promoting sequence, which can promote the efficient expression of the fusion protein in a heterologous host, and is conducive to industrial fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0059] Figure 1 is the plasmid map of the recombinant plasmid pPIC9k-C1701-DSG1 in Example 1 of the present invention;

[0060] Figure 2 is the plasmid map of the recombinant plasmid pPIC9k-4C3L-C1701-DSG1 in Example 1 of the present invention;

[0061] Figure 3 This is a diagram showing the SDS-PAGE electrophoresis results of the shake flask fermentation supernatants of the recombinant bacteria GS115 / pPIC9K-4C3L-C1701-DSG1-4 and the induced expression of the recombinant bacteria GS115 / pPIC9K-C1701-DSG1-4 in Example 1 of the present invention;

[0062] Figure 4 This is an SDS-PAGE electrophoresis result of the high-density fermentation supernatant of the recombinant bacteria GS115 / pPIC9K-4C3L-C1701-DSG1-4 in Example 1 of the present invention;

[0063] Figure 5 This is a diagram showing the purification results of the high-density fermentation broth of the recombinant bacteria GS115 / pPIC9K-4C3L-C1701-DSG1-4 in Example 1 of the present invention;

[0064] Figure 6 is the result of promoting cell proliferation activity of each group in Experimental Example 1 of the present invention;

[0065] Figure 7 is the result of promoting cell adhesion in each group in Experimental Example 1 of the present invention;

[0066] Figure 8 These are the observation results of promoting cell migration in each group in Experimental Example 1 of the present invention;

[0067] Figure 9 This is the statistical result of the migration rate of cells promoted in each group in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0068] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0069] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0070] The gene sequences involved in the following examples were all synthesized by GenScript Biotech Co., Ltd.

[0071] Example 1 Fusion Protein

[0072] This embodiment provides a method for preparing a fusion protein, comprising the following steps:

[0073] 1. Obtaining fusion protein recombinant plasmid

[0074] Obtaining the target fusion protein: In this example, the functional region fragment sequence of type XVII collagen was screened out as follows:

[0075] C1701 (SEQ ID No. 1):

[0076] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGP MGQRGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQAGPPGH PGPPGPRGHKGEKGDKGDQ.

[0077] The functional region fragment sequence of desmoglein (Dsg) screened out in this example is as follows:

[0078] DSG1-4 (SEQ ID No. 2):

[0079] NMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVT KEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQ.

[0080] In this example, in order to promote the expression of proteins in Pichia pastoris, a type III collagen expression-promoting sequence (SEQ ID No. 3) was screened.

[0081] SEQ ID No.3:

[0082] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPA GERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKG PAGERGAP.

[0083] Target fusion protein: The above two sequences (SEQ ID No. 1 and SEQ ID No. 2) and three sequences (SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3) were connected through three GGGGS flexible linkers to obtain the target fusion protein sequences C1701-DSG1 (SEQ ID No. 4) and 4C3L-C1701-DSG1 (SEQ ID No. 5).

[0084] C1701-DSG1-4 (SEQ ID No. 4):

[0085] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGPPGQKGEMGT PGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ GGGGSGGGGSGGGGS NMGSNDKVGDFVATDLDTGRPSTTVRY VMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQ.

[0086] The predicted isoelectric point / molecular weight (pI / Mw) is: 9.35 / 20.2 KD.

[0087] The nucleotide sequence encoding SEQ ID No.4 is as follows (SEQ ID No.6):

[0088] GGGTCCCCCGGTCCAAAGGGAGATATGGGCAGTCCAGGCCCTAAAGGGGACCGCGGCTTCCCTGGAACCCCCGGTATTCCAGGGCCGTTGGGACACCCGGGTCCACAGGGTCCTAAAGGTCAAAAAGGTTCAGTCGGGGACCCGGGAATGGAAGGGCCTATGGGACAGAGGGGGCCGCCCGGCCAGAAAGGCGAGATGGGTACTCCAGGACCTAAAGGCGATCGTGGGCCCGCCGGTCCCCCAGGGCATCCGGGCCCGCCTGGGCCACGGGGACATAAGGGGGAGAAGGGTGATAAGGGAGACCAAGGAGGAGGAGGTTCGGGGGGTGGGGGGAGCGGCGGAGGCGGTTCAAATATGGGCTCTAACGATAAGGTCGGCGACTTTGTAGCGACTGATTTAGACACGGGAAGGCCCTCCACGACAGTGAGATACGTAATGGGGAACAATCCTGCAGATCTGTTAGCTGTTGACTCGAGAACCGGTAAGCTTACCCTCAAAAACAAGGTTACGAAAGAACAGTATAACATGCTAGGCGGCAAATACCAAGGCACGATATTGTCTATCGATGACAATCTGCAACGAACTTGTACAGGAACAATTAACATAAATATCCAG.

[0089] 4C3L-C1701-DSG1-4 (SEQ ID No.5):

[0090] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGGGGSGGGGSGGGGSEF GSPGPKGDM GSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGPPGQKGEMGTPPGPKGDRGPAGPPGHPGP PGPRGHKGEKGDKGDQGGGGSGGGGSGGGGS NMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKL TLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQ

[0091] The predicted isoelectric point / molecular weight (pI / Mw) is: 9.59 / 33KD.

[0092] The nucleotide sequence encoding SEQ ID No. 5 is as follows (SEQ ID No. 7):

[0093]

[0094] A terminator is added to the C-terminus of the gene encoding the amino acid sequence of the target fusion protein, and the DNA sequence encoding the target fusion protein is optimized for Pichia pastoris preference, so that the target fusion protein is more suitable for expression in Pichia pastoris. The optimized base sequence of the target fusion protein is entrusted to GenScript Biotech Co., Ltd. for gene fragment synthesis (the synthesized sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively). The synthesized gene fragment is inserted into the pPIC9K plasmid through the EcoRⅠ and NotⅠ restriction sites to obtain recombinant desmosome-ⅩⅦ collagen fusion protein plasmids pPIC9K-C1701-DSG1-4 and pPIC9K-4C3L-C1701-DSG1-4, respectively. The plasmid maps are shown in FIG. Figure 1 and Figure 2 As shown, the nucleotide sequence of pPIC9K-4C3L-C1701-DSG1-4 is shown in SEQ ID NO.10, and the nucleotide sequence of pPIC9K-C1701-DSG1-4 is the sequence shown in SEQ ID NO.10, wherein the corresponding nucleotide sequence of 4C3L-C1701-DSG1-4 is replaced by the nucleotide sequence of C1701-DSG1-4.

[0095] 2. Construction of expression plasmid

[0096] 2.1 Plasmid linearization

[0097] The plasmids pPIC9K-C1701-DSG1-4 and pPIC9K-4C3L-C1701-DSG1-4 were linearized using QuickCut SacⅠ at 37°C for 5 h. The enzyme digestion system was as follows:

[0098] Table 1. Enzyme digestion system

[0099] Reagents Volume (μl) plasmids 10-15 μg Quick Cut Sac I (commercially available) 10 10×Quick Cut buffer (commercially available) 5 Total volume 50

[0100] After digestion, add 0.1 volumes of 3M NaAc (pH 5.2) and 2.5 volumes of anhydrous ethanol and incubate at -20°C overnight. Centrifuge at 13,000 rpm for 20 minutes at 4°C and discard the supernatant. Rinse with 700 μl of 75% ethanol and centrifuge at 13,000 rpm for 20 minutes, discarding the supernatant. Repeat this process. Invert the EP tube onto absorbent paper on a clean bench for approximately 10 minutes to remove as much water and residual ethanol as possible. Redissolve the plasmid in 20 μl of ddH2O, dilute 10-fold with 1 μl, and assay the nucleic acid concentration using a one-drop assay.

[0101] 2.2 Preparation of competent yeast cells

[0102] After streaking the Pichia pastoris host strain GS115, a single colony was picked and inoculated into 20 ml of YPD and cultured at 30°C and 225 rpm for 24 h; the colony was transferred to 50 ml of YPD liquid medium at a ratio of 1:1000 and cultured at 30°C and 225 rpm until the OD 600nm＝ 1.3-1.5; Transfer the bacterial solution into a sterile 50ml centrifuge tube, centrifuge at 4℃, 1500rpm for 5min, discard the supernatant and collect the bacteria; resuspend the bacterial pellet with 50ml of pre-cooled sterile ultrapure water, centrifuge at 4℃, 1500rpm for 5min; discard the supernatant, and resuspend the bacterial pellet with 50ml of pre-cooled sterile ultrapure water; centrifuge at 4℃, 1500rpm for 5min, discard the supernatant, and resuspend the cell pellet with 40ml of pre-cooled sterile 1M (mol / L) sorbitol; centrifuge at 4℃, 1500rpm for 5min, discard the supernatant, and resuspend the cell pellet with 100-150μl of pre-cooled sterile 1M sorbitol, gently rotate to mix, and place on ice for use.

[0103] 2.3 Electrotransformation into Pichia pastoris GS115 competent cells

[0104] Take 100μl of Pichia pastoris GS115 competent cells from step 2.2 and mix with 10μl of linearized plasmid obtained in step 2.1, transfer to a pre-cooled electroporation cuvette, and immediately place on ice for 5 minutes. Select the yeast mode of the electroporator and perform electroporation. Then immediately add 1mL of pre-cooled 1M sorbitol to the electroporation cuvette, mix well, transfer the mixture to a sterile EP tube, and incubate in a 30℃ incubator for 1-2 hours. Take 100μl~200μl of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 10 minutes, and incubate it upside down in a 30℃ incubator for about 2~5 days until a single colony appears.

[0105] 2.4 Screening of positive transformants

[0106] Add 2 mL of sterile double-distilled water to the surface of the MD plate, and then gently scrape off the His on the surface of the plate with a sterile triangular spreader. + The transformants were transferred to 50 mL centrifuge tubes. The colonies were counted and diluted to 10 with sterile water. 5A cell suspension at a concentration of 10 cells / mL is spread onto a YPD plate containing 0.5 mg / mL G418 (Geneticin 418), inverted, and incubated at 30°C for 3-4 days until a single colony appears. A single colony is picked from the YPD plate and transferred to a 96-well plate containing 200 μL of YPD medium and continued to be incubated at 30°C. After 48 hours, the bacterial suspension is agitated and 10 μL is transferred from each well to a new 96-well plate containing 190 μL of YPD. Incubate for another 24 hours, then repeat the above process. After 24 hours, the bacterial suspension in a third 96-well plate is agitated and 1 μL is aspirated and applied to YPD plates containing 1 mg / mL, 2 mg / mL, and 4 mg / mL G418, respectively, for further incubation. Transformants that can grow on the plate containing the high concentration of 4 mg / mL G418 indicate that they contain multiple copies of the target gene. This screening step can identify engineered recombinant yeast strains capable of highly expressing the gene.

[0107] 2.5 Identification of positive transformants

[0108] Take 50 μL of the remaining bacterial solution from the first 96-well plate in step 2.4 above, boil it in a boiling water bath for 10 min, freeze it in liquid nitrogen for 30 min, boil it in a boiling water bath for 10 min, repeat freeze-thaw cycles, centrifuge it at 12,000 rpm for 5 min, and use the supernatant as a PCR template to determine whether the target gene has been integrated into the yeast chromosome.

[0109] Upstream primer: 5'AOX1 (5'-GACTGGTTCCAATTGACAAGC-3') (SEQ ID NO. 8);

[0110] Downstream primer: 3'AOX1 (5'-GCAAATGGCATTCTGACATCC-3') (SEQ ID NO. 9);

[0111] PCR amplification system:

[0112] Table 2. PCR amplification system

[0113]

[0114] PCR amplification conditions: pre-denaturation at 98°C for 5 min, thermal denaturation at 98°C for 50 s, annealing at 60°C for 1 min, extension at 72°C for 60 s, 35 cycles; annealing at 72°C for 10 min.

[0115] The amplified products were subjected to 1.5% agarose gel electrophoresis to identify whether the gene fragment of the expected size was amplified. The transformants with the gene fragment of the expected size were identified as positive transformants and were recorded as GS115 / pPIC9K-4C3L-C1701-DSG1-4 and GS115 / pPIC9K-C1701-DSG1-4, respectively.

[0116] 3. Induced Expression of Recombinant Yeast

[0117] The culture medium formula used is as follows:

[0118] 1) YPD complete medium:

[0119] Yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L (solid medium containing 2% agar);

[0120] 2)MD solid medium:

[0121] YNB without amino acids, nitrogen source 13.4 g / L; 0.4 mg / L biotin; 20 g / L glucose (solid medium containing 2% agar);

[0122] 3) BMGY yeast growth medium:

[0123] Yeast extract 10g / L, peptone 20g / L, 3g / L K2HPO4, 11.8g / L KH2PO4, add water to 890mL, sterilize at 121℃ for 20 minutes, then add 100mL of 10×YNB (13.4g / L), 1mL of 500×biotin (4×10 -4 g / L), glycerol 10mL.

[0124] 4) BMMY yeast induction medium:

[0125] Yeast extract 10g / L, peptone 20g / L, 3g / L K2HPO4, 11.8g / L KH2PO4, add water to 895mL, sterilize at 121℃ for 20 minutes, then add 100×YNB 100mL (13.4g / L), 500×biotin 1mL (4×10 -4 g / L), methanol 5mL.

[0126] The specific operation is as follows: pick a single colony of the positive transformant identified in 2.4 and inoculate it into BMGY medium, and culture it at 30℃ and 220rpm for 24h until the OD 600nm =2-6. According to the measured OD 600nmAdjust the volume of BMGY culture solution to the value, collect the cells at 3000rpm for 10min, and resuspend the cells with BMMY culture medium of the same volume as BMGY to make the initial OD 600nm The value was 2.0. The culture was continued at 30℃ and 220rpm. 0.5v / v% methanol was added to the culture medium every 24h. The bacterial liquid samples were taken 24h, 48h, 72h, and 96h after induction, and the expression supernatant was collected by centrifugation. The expression level of the target protein was analyzed by SDS-PAGE electrophoresis ( Figure 3 ).

[0127] Analysis of results: SDS-PAGE electrophoresis showed that the expression level of C1701-DSG1-4 was low, and clear bands could be seen after concentration. However, the expression level of 4C3L-C1701-DSG1-4 with the addition of expression-promoting sequences was significantly higher than the former, and obvious electrophoresis bands could be detected within 24 hours.

[0128] 4. Fermentation in fermenter

[0129] Seed medium: YPD (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L);

[0130] Basal fermentation medium (BSM): 85wt% H3PO4 26.7mL / L; CaSO4·2H2O 1.86g / L; K2SO4 20g / L;

[0131] MgSO4·7H2O 16 g / L; KOH 4 g / L; glycerol 35.0 g / L; PTM1 5 mL / L; biotin (500×) 6.35 mL / L.

[0132] The PTM1 solution was prepared according to the Invitrogen operating manual. The specific formula was as follows: CuSO4·5H2O 6.0 g / L; NaI 0.08 g / L; MnSO4·H2O 3.0 g / L; NaMoO4·2H2O 0.2 g / L; H3BO3 0.02 g / L; CoCl2 0.5 g / L; ZnCl2 20.0 g / L; FeSO4·7H2O 65.0 g / L; biotin 0.2 g / L; H2SO4 5.0 mL / L. The solution was sterilized by filtration through a 0.22 μm filter membrane and stored at room temperature.

[0133] Specific implementation methods:

[0134] (1) The recombinant bacteria (GS115 / pPIC9K-4C3L-C1701-DSG1-4) were activated and inoculated into YPD medium for amplification to obtain seed solution;

[0135] (2) The seed liquid was added to a 30 L fermentation tank containing 10 to 15 L of fermentation medium (BSM) at an inoculum size of 8 to 10%, with an initial stirring speed of 200 to 400 rpm and a tank pressure of 0.03 to 0.05 MPa. The pH was controlled at 5.0 to 6.0 and the temperature was 25 to 30°C. The air flow rate and speed were adjusted to control the dissolved oxygen at about 25 to 35%. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol (50% glycerol aqueous solution by mass) was added to supplement the carbon source.

[0136] (3) When the wet weight increases to about 180-200 g / L, stop feeding glycerol, starve the culture for 1-2 h, and start feeding methanol after the glycerol is exhausted, entering the methanol induction stage. Use ammonia water to adjust the pH to 5.0-6.0, adjust the rotation speed, air flow rate and feeding rate of methanol to control the dissolved oxygen at about 25-35%, and end the fermentation after 72 h of induction;

[0137] (4) The fermentation product was centrifuged at 9000 rpm for 30 min to separate the solid and liquid to obtain the fermentation supernatant. The supernatant was ultrafiltered and concentrated using a hollow fiber ultrafiltration system with a molecular weight cutoff of 10.0 KD. The retentate was collected to obtain the crude extract of the fusion protein. The yield of the protein solution was about 1.8-2.0 g / L. The electrophoresis results of the samples taken during the fermentation process were as follows: Figure 4 shown.

[0138] 6 Purification

[0139] The culture supernatant was collected by centrifugation.

[0140] Use a cation exchange medium (chromatographic filler is SP Purose 6 High Performance produced by Qianchun, loaded on a GE Akta chromatography system), and balance the chromatography column with citric acid-sodium citrate buffer (50mM, pH 5.0) until the conductivity value and A280 absorbance value remain unchanged. Set the sample loading flow rate to 5-10mL / min, detect the UV A280 absorbance value, and start sampling when it rises. After the loading is completed, balance the cationic chromatography medium with citric acid-sodium citrate buffer until the UV and conductivity drop to the lowest and no longer change, and stop sampling. Then, gradient elution is performed with citric acid-sodium citrate buffer containing NaCl (1M), and the corresponding purified protein (eluate), flow-through, and impurities are collected for electrophoresis, see Figure 5 After ultrafiltration and liquid exchange, recombinant desmosome-type XVII collagen fusion protein (4C3L-C1701-DSG1) was obtained.

[0141] Experimental Example 1 Cell proliferation test

[0142] Refer to Appendix 3528 of the 2020 edition (Part III) of the Chinese Pharmacopoeia. This assay is based on the finding that recombinant proteins stimulate the growth of mouse embryonic fibroblasts (BALB / c 3T3 cells). The growth of BALB / c 3T3 cells varies depending on the biological activity of different recombinant proteins, thereby detecting the biological activity of promoting cell proliferation in vitro.

[0143] Test materials:

[0144] Complete cell culture medium: Add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C.

[0145] Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C.

[0146] Digestion solution: 0.25% trypsin.

[0147] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up to 1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

[0148] Thiazolyl blue (MTT) solution: Weigh 0.10 g of MTT powder and dissolve in 20 ml of PBS. Sterilize by filtering through a 0.22 μm filter. Store at 4°C in the dark.

[0149] Maintenance medium: serum-free medium containing 0.4%;

[0150] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0151] Samples: Recombinant desmosome-type XVII collagen fusion protein (4C3L-C1701-DSG1 prepared in Example 1), diluted to 1 mg / mL; recombinant humanized type XVII collagen (COLXVII, purchased from Aladdin, catalog number rp212899), dissolved and diluted to 1 mg / mL; recombinant desmosomal protein (DSG1, purchased from Apti, catalog number PA1000-9347), dissolved and diluted to 1 mg / mL.

[0152] Specific implementation method: BALB / c 3T3 cell line was cultured in complete culture medium at 37°C and 5% carbon dioxide, and the cell density was controlled to be 2.0×10 5 cells / ml, and used for biological activity determination 24 to 36 hours after passage. Discard the culture medium in the culture flask, digest and collect the cells, and use complete culture medium to make 5.0×10 5A cell suspension of 100 μl cells / ml was plated in a 96-well cell culture plate, with 100 μl per well, and cultured at 37°C and 5% CO2. After 24 hours, the plate was replaced with maintenance medium and incubated at 37°C and 5% CO2 for another 24 hours. The maintenance medium was discarded from the prepared cell culture plate, and 100 μl of the standard solution (refer to Chinese Pharmacopoeia, Part III, 3528; the standard is human epidermal growth factor) and the test sample (the aforementioned sample) solution were added to each well, with duplicate wells for each sample. The plates were incubated at 37°C and 5% CO2 for 72 hours. Twenty μl of MTT solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 5 hours. All procedures were performed under sterile conditions. After discarding the liquid from the plate, 100 μl of DMSO was added to each well, mixed, and the absorbance was measured at 570 nm using a microplate reader with a reference wavelength of 630 nm. The results were recorded.

[0153] Data processing: The test data are processed using a computer program or four-parameter regression method, and the results are calculated according to the following formula:

[0154] Corrected titer (U / ml) = Pr × Ds × Es / Dr × Er

[0155] Where Pr is the biological activity of the standard, U / ml;

[0156] Ds is the pre-dilution multiple of the test sample;

[0157] Es is the dilution multiple of the test sample equivalent to the half-effectiveness of the standard sample;

[0158] Dr is the pre-dilution multiple of the standard;

[0159] Er is the dilution multiple of the half-effective dose of the standard;

[0160] The results are as follows Figure 6 As shown in the figure, according to the specific activity results, the specific activity of the commercial recombinant humanized type XVII collagen in promoting 3T3 cell proliferation is about 180 U / mg, the specific activity of the recombinant desmosome DSG1 protein in promoting 3T3 cell proliferation is about 380 U / mg, and the specific activity of the recombinant desmosome-type XVII collagen fusion protein in promoting 3T3 cell proliferation is 859 U / mg, which is significantly higher than the former recombinant humanized type XVII collagen and recombinant desmosome DSG1 protein. In the figure, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0161] Experimental Example 2 Cell Adhesion Promotion Test

[0162] Test materials:

[0163] Complete cell culture medium: Add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C.

[0164] Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C.

[0165] Digestion solution: 0.25% trypsin.

[0166] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up to 1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

[0167] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0168] BSA (purchased from Sigma).

[0169] Samples: Recombinant desmosome-type XVII collagen fusion protein (4C3L-C1701-DSG1 prepared in Example 1), diluted to 1 mg / mL; recombinant humanized type XVII collagen (COLXVII, purchased from Aladdin, catalog number rp212899), dissolved and diluted to 1 mg / mL; recombinant desmosomal protein (DSG1, purchased from Apti, catalog number PA1000-9347), dissolved and diluted to 1 mg / mL.

[0170] Specific implementation method: The sample was pre-diluted to 0.5 μg / ml with PBS. After pre-dilution, a 2-fold serial dilution was performed in a 96-well plate, making a total of 8 dilutions. 50 μl of sample of different dilutions was added to each well. Each concentration was repeated in triplicate. A negative control (no sample) was set up, and 50 μl of PBS was added as a control. The plates were incubated at 4°C overnight. After incubation, the liquid in the plate was discarded, and 100 μl of PBS was added to each well for washing three times. After washing, 100 μl of 30 μg / μL BSA was added to each well for blocking, and the plates were incubated at 37°C for 1 hour. After incubation, the liquid in the plate was discarded, and 100 μl of PBS was added to each well for washing three times. Then, a fibroblast suspension (BALB / c 3T3 cells) was added, and the cell seeding density was 5.0×10 5 cells / ml, inoculate 100 μl per well, and incubate in an incubator for 5 h. Wash the plate three times with PBS after incubation. Observe cell adhesion under a microscope and count the number of adherent cells at five points, excluding the edge, under a 200x microscope. Calculate the titer based on the curve fitted with the counting results.

[0171] Data processing: The experimental data were processed by four-parameter regression calculation using a computer program or ELISACalc software to obtain the formula: Y = (ad) / [1+(x / c)b]+d. The data points of X and Y were collected or generated, where X is the gradient dilution factor (1, 2, 4, 8, 16, 32, 64, 128) or converted into the corresponding protein concentration (mg / mL or μg / mL), and Y is the average number of cells per well corresponding to X. 2 Statistical indicators such as σ2 and σ3 values ​​were used to evaluate the fitting effect and ensure the goodness of model fitting.

[0172] Where: a is the estimated value of the asymptote on the curve;

[0173] b is the slope of the curve;

[0174] c is the dose corresponding to half of the maximum binding (i.e., half-effective concentration);

[0175] d is the estimated value of the asymptote under the curve;

[0176] Then, the dilution factor is raised to the power of c (i.e. 2 c ) value was calculated to obtain the titer U / ml, which was then divided by the corresponding protein concentration to obtain the specific activity U / mg.

[0177] The test results are as follows Figure 7 As shown, commercial type XVII collagen had no significant cell adhesion promoting activity. Recombinant desmosomal DSG1 protein had a specific activity of approximately 157 U / mg. Recombinant desmosomal-type XVII collagen fusion protein exhibited some cell adhesion promoting activity, with a specific activity of approximately 662 U / mg, significantly higher than that of commercial type XVII collagen and recombinant desmosomal DSG1 protein. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0178] Experimental Example 3 Cell migration promotion test (cell scratch test)

[0179] Refer to Appendix C, "Cell Scratch Method," of the industry standard "YY / T 1849-2022." When cells grow to a confluent monolayer, a blank area, called a "scratch," is artificially created on the confluent monolayer. Cells at the edge of the scratch gradually migrate into the blank area, healing the scratch. Images are captured at the beginning and at regular intervals during cell migration, and the images are compared to determine the cell migration rate.

[0180] Samples: Recombinant desmosome-type XVII collagen fusion protein (4C3L-C1701-DSG1 prepared in Example 1), diluted to 1 mg / mL; recombinant humanized type XVII collagen (COLXVII, purchased from Aladdin, catalog number rp212899), dissolved and diluted to 1 mg / mL; recombinant desmosomal protein (DSG1, purchased from Apti, catalog number PA1000-9347), dissolved and diluted to 1 mg / mL.

[0181] HaCat immortalized human keratinocytes (provided by Anhui Medical University).

[0182] Specific implementation method: First, use a marker pen to align with a ruler on the back of a 6-well plate and draw horizontal lines evenly, about every 0.5 to 1 cm, across the holes. Add a concentration of 5×10 5 cells / ml of HaCat cell suspension. The next day, it was observed that all the cells in the 6-well plate had grown into a single layer. Use the tip of the gun to measure the ruler and scratch perpendicularly to the horizontal line on the back, with 2 scratches in each well. Wash the cells 3 times with PBS to wash off the suspended cells. According to the grouping, add 1.8ml of serum-free culture medium to the wells, then add 200μl of sample, and add an equal amount of PBS solution to the cell control wells. Make 3 replicates for each group. Place in a 37℃, 5% CO2 incubator for culture. Record and take pictures of the fixed positions during subsequent observations.

[0183] Data processing: The scratch area was measured, and the migration rate of each group of cells was calculated by dividing the total area of ​​migrating cells in the fixed scratch area by the initial area of ​​the fixed scratch area.

[0184] The results are as follows Figure 8 and Figure 9 As shown, the experimental groups all showed significant cell migration-promoting activity compared to the cell control group. The recombinant desmosome-type XVII collagen fusion protein promoted cell migration by approximately 38.8%, while the commercial XVII collagen promoted cell migration by 37.8%, and the recombinant desmosome DSG1 protein promoted cell migration by approximately 35.7%. The cell migration-promoting activities of the three recombinant proteins were basically the same, with no significant differences.

[0185] Experimental Example 4 Cytotoxicity Test

[0186] Refer to Medical Device Biological Evaluation Part 5: In Vitro Cytotoxicity Tests (GB / T 16886.5-2017). Cytotoxicity is measured by observing cell morphology and the MTT assay, and by quantifying the number of live and dead cells in the sample and measuring cell metabolic activity.

[0187] Test materials:

[0188] Complete cell culture medium: DMEM culture medium (containing double antibodies) with 10% fetal bovine serum added and stored at 4°C.

[0189] Serum-free culture medium: DMEM culture medium (containing double antibodies), stored at 4°C.

[0190] Digestion solution: 0.25% trypsin.

[0191] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up to 1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

[0192] Thiazolyl blue (MTT) solution: Weigh 0.10 g of MTT powder and dissolve in 20 ml of PBS. Sterilize by filtering through a 0.22 μm filter. Store at 4°C in the dark.

[0193] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0194] Sample: recombinant fibronectin-type XVII collagen fusion protein (COLXVII-FN, C1701-FN prepared in Example 1), diluted to 1 mg / mL.

[0195] Specific implementation method: BALBIC mouse embryonic fibroblasts were cultured in complete culture medium at 37°C and 5% CO2, and the cell density was controlled at 1.0×10 5 cells / mL, discard the culture medium in the culture flask 24 to 36 hours after passage. After digestion, collect the cells with complete culture medium and make 5.0×10 5 cells / mL cell suspension. 100 μL of the cell suspension was inoculated into each well of a 96-well plate and cultured for 24 hours. After the cells had grown into a monolayer, the original culture medium was aspirated. 100 μL of the test sample dilutions (samples were serially diluted 10-fold in complete cell culture medium) at different concentrations, a blank control (complete cell culture medium), and a negative control (PBS) were added to the experimental groups. Six replicate wells were prepared for each group except the experimental groups. Within the experimental groups, three replicate wells were prepared for each concentration of each sample. The plates were incubated at 37°C, 5% CO2. After 24 hours, the 96-well plates were removed and cell morphology was observed under a microscope. The liquid was then aspirated and 50 μL of MTT (1 mg / mL) was added to each well. The plates were incubated in a CO2 incubator for 2 hours. The MTT solution was discarded and 100 μL of DMSO solution was added to each well. The plates were shaken and the absorbance was measured at 570 nm (reference wavelength 650 nm) on a microplate reader. Calculate cell survival rate. When the cell survival rate is lower than that of the blank control group, the sample is cytotoxic. Otherwise, it is non-cytotoxic. Statistical method: mean ± standard deviation (ˋx±s); survival rate (%) = (OD570 Mean / blank control group OD 570 mean)×100%.

[0196] Results: Compared with the blank control group, the relative cell viability was >90%, with no significant difference (P>0.05). Cell morphology remained unchanged compared with the blank control group. The highest dose group, with an average cell viability exceeding 90%, was selected, and the results showed no significant cytotoxicity at doses ranging from 10 to 100 μg / ml.

[0197] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant type XVII collagen fusion protein, characterized in that: include: At least one functional domain fragment of type XVII collagen, at least one functional domain fragment of desmosome core glycoprotein.

2. The recombinant type XVII collagen fusion protein according to claim 2, characterized in that The type VII collagen functional region fragment is selected from any one of the following amino acid sequences (1)-(2): (1) having an amino acid sequence as shown in any one of SEQ ID NO. 1; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence represented by any one of SEQ ID NO. 1 described in (1).

3. The recombinant type XVII collagen fusion protein according to claim 1 or 2, characterized in that The functional region fragment of the core glycoprotein of the desmosome is selected from any one of the following amino acid sequences (1)-(2): (1) having the amino acid sequence shown in SEQ ID NO. 2; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence shown in SEQ ID NO. 2 described in (1).

4. The recombinant type XVII collagen fusion protein according to any one of claims 1 to 3, characterized in that The recombinant type XVII collagen fusion protein is also fused with a type III collagen expression-promoting sequence; Optionally, the type III collagen expression-promoting sequence is selected from any one of the following amino acid sequences (1)-(2): (1) having the amino acid sequence shown in SEQ ID NO. 3; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence shown in SEQ ID NO. 3 described in (1).

5. The recombinant type XVII collagen fusion protein according to claim 4, characterized in that The C-terminus of the functional region fragment of type XVII collagen is fused with the functional region fragment of the core glycoprotein of the desmosome; Optionally, the N-terminus of the type XVII collagen functional region fragment is fused with the type III collagen expression-promoting sequence; Optionally, the functional domain fragment of type XVII collagen and the functional domain fragment of the desmosome core glycoprotein are connected by at least one linker; preferably, the number of linkers is 1-3; Optionally, the type III collagen expression-promoting sequence and the type XVII collagen functional region fragment are connected by at least one linker; preferably, the number of linkers is 1-3; Optionally, the amino acid sequence of the linker is GGGGS; Optionally, the recombinant type XVII collagen fusion protein is selected from any one of the following amino acid sequences (1)-(2): (1) having an amino acid sequence as shown in SEQ ID NO.4 to SEQ ID NO.5; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence shown in SEQ ID NO. 4 to SEQ ID NO. 5 described in (1).

6. A biomaterial, characterized in that Includes any of the following: 1) A nucleic acid molecule encoding the recombinant type XVII collagen fusion protein according to any one of claims 1 to 5; optionally, the nucleic acid molecule is DNA or RNA; 2) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line expressing the recombinant type XVII collagen fusion protein according to any one of claims 1 to 5; 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in 1); 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in 2) or 3); 5) A host cell containing the recombinant vector described in 2) or 3) or 4).

7. The biomaterial according to claim 6, characterized in that Including: the initial plasmid vector of the recombinant vector is pPIC9K plasmid; and / or, the host cell comprises yeast; And / or, the method for constructing the recombinant vector comprises: inserting a nucleic acid molecule encoding the recombinant type XVII collagen fusion protein into a plasmid vector through an enzyme cleavage site to obtain the recombinant vector; And / or, the method for constructing the host cell containing the recombinant vector comprises: linearizing the recombinant vector by enzyme digestion, then transferring the recombinant vector into competent host cells, culturing, and screening positive transformants.

8. A method for preparing the recombinant type XVII collagen fusion protein according to any one of claims 1 to 5, characterized in that: include: Synthesizing a gene encoding the recombinant type XVII collagen fusion protein according to any one of claims 1 to 5; constructing a recombinant vector containing the encoding gene; The recombinant vector is transformed into host cells and fermented and cultured.

9. The preparation method according to claim 8, characterized in that The initial plasmid vector of the recombinant vector is pPIC9K plasmid; and / or, the host cell comprises yeast; And / or, the fermentation culture conditions include any one of the following: The inoculation rate is 8% to 10%; Stirring speed 200-400 rpm; Tank pressure 0.03~0.05MPa; Control pH 5.0-6.0; Temperature 25-30℃; Dissolved oxygen is controlled at 25-35%; And / or, after the fermentation culture reaches a set value, methanol is added for induction culture, the pH is adjusted to 5.0-6.0, the dissolved oxygen is controlled at 25-35%, and the induction is carried out for 48-72 hours.

10. A use of the recombinant type XVII collagen fusion protein according to any one of claims 1 to 5, the biomaterial according to claim 6 or 7, or the recombinant type XVII collagen fusion protein prepared by the preparation method according to claim 8 or 9, comprising any of the following: (1) Promoting cell proliferation or preparing products that promote cell proliferation; (2) promoting cell adhesion or preparing products that promote cell adhesion; (3) Promoting cell migration or preparing products that promote cell migration; (4) Preparation of skin care products or cosmetics; (5) Preparation of biomedical materials; Optionally, the product includes skin care products, cosmetics, medicines or biomedical materials.

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