Recombinant XVII humanized collagen and preparation method thereof

Abstract

The embodiment of the invention discloses recombinant XVII humanized collagen and a preparation method thereof, relates to the technical field of recombinant protein, and aims to solve the technical problem of low preparation efficiency of existing XVII type collagen. The preparation method of the recombinant XVII humanized collagen comprises the following steps: obtaining an amino acid sequence containing an integrin receptor site "RGD" and a binding site "KGHK"; based on the amino acid sequence, obtaining recombinant humanized collagen protein granules; the method comprises the following steps: obtaining recombinant lysine hydroxylase gene plasmids on the basis of a human gene LH2; and obtaining the recombinant XVII humanized collagen on the basis of the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid.

Description

Technical Field

[0001] This application relates to the field of recombinant protein technology, specifically to a recombinant XVII humanized collagen and its preparation method. Background Technology

[0002] Type XVII collagen is an important component of many human tissues. It is a transmembrane protein found in the skin, particularly in the basement membrane zone at the junction of the epidermis and dermis. It plays a crucial role in maintaining the integrity of skin structure and promoting skin repair and regeneration. However, type XVII collagen is present in very small amounts in humans and animals, making its extraction extremely difficult. Its long amino acid chains and large molecular weight make extraction or synthesis very challenging and prevent mass production. Therefore, there are currently very few similar raw materials and products on the market.

[0003] Researchers have attempted to produce type XVII collagen using various recombinant expression systems, including insect cells, mammalian cells, and bacteria. However, these systems have limitations in expression efficiency, proper protein modification, and feasibility for large-scale production. Furthermore, maintaining the protein's native structure, biological activity, and in vitro stability presents challenges. These technical challenges result in low efficiency during the actual preparation of type XVII collagen. Summary of the Invention

[0004] The main objective of this application is to provide a recombinant XVII humanized collagen and its preparation method, aiming to solve the technical problem of low preparation efficiency of existing XVII type collagen.

[0005] To achieve the above objectives, the technical solutions adopted in the embodiments of this application are as follows: In a first aspect, embodiments of this application provide a method for preparing recombinant XVII humanized collagen, comprising the following steps: Obtain the amino acid sequence containing the integrin receptor site “RGD” and the binding site “KGHK”; Based on the amino acid sequence, recombinant humanized collagen protein particles were obtained; Based on the human gene LH2, a recombinant lysine hydroxylase gene plasmid was obtained. Recombinant XVII humanized collagen was obtained based on the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid.

[0006] As some optional embodiments of this application, obtaining the amino acid sequence containing the integrin receptor site "RGD" and the binding site "KGHK" includes: Protein analysis was performed on the triple helix region of the mature peptide derived from human type XVII collagen. The C1 amino acid sequence shown in SEQ ID No. 1, the C2 amino acid sequence shown in SEQ ID No. 2, and the C3 amino acid sequence shown in SEQ ID No. 3 of the human type XVII collagen peptide domain fragment were selected respectively. The C1 amino acid sequence and the C2 amino acid sequence were connected end to end to form the C4 collagen peptide shown in SEQ ID No. 4. The integrin receptor site "RGD" was obtained from the C4 amino acid sequence. The C4 collagen peptide is linked end-to-end with the C3 amino acid sequence to form the C5 collagen peptide as shown in SEQ ID No. 5, and the skin hair follicle repair binding site "KGHK" is obtained from the C5 collagen peptide.

[0007] As some optional embodiments of this application, obtaining recombinant humanized collagen protein particles based on the amino acid sequence includes: After repeating the C5 collagen peptide tandemly 7 times, the sequence shown in SEQ ID No. 6 was obtained. A terminator was added to its C-terminus, and the DNA sequence encoding the protein was optimized by Pichia pastoris preference design to obtain the optimized base sequence shown in SEQ ID No. 7. After adding the base sequence AAAAGA to the 5' end of the optimized base sequence, gene fragment synthesis was performed. The synthesized gene fragment was then processed... EcoR I and Not The restriction enzyme site I was inserted into the pPICZαA plasmid to obtain recombinant humanized collagen protein plasmid.

[0008] As some optional embodiments of this application, obtaining the recombinant lysine hydroxylase gene plasmid based on the human LH2 gene includes: The human gene LH2 with an amino acid sequence such as SEQ ID No. 8 was optimized by adding 6×His and a terminator to the C-terminus, resulting in the optimized DNA sequence such as SEQ ID No. 9. The optimized DNA sequence was used to synthesize gene fragments, and the fragments were inserted into the pPIC3.5K plasmid through the EcoRI and NotRI restriction sites to obtain the recombinant lysine hydroxylase gene plasmid.

[0009] As some optional embodiments of this application, obtaining recombinant XVII humanized collagen based on the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid includes: Gene fragments were synthesized from the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid to obtain pPICZαA-COL plasmid and pPIC3.5K-rhLH2 plasmid, respectively. After linearizing the pPICZαA-COL plasmid, it was electroporated into GS115 competent cells to obtain multiple transformants; after screening and identification of the multiple transformants, positive transformants were obtained; based on the positive transformants, recombinant engineered bacterial competent cells were prepared. After linearizing the pPIC3.5K-rhLH2 plasmid, it was electroporated into the competent cells of the recombinant engineered bacteria to obtain multiple secondary transformants; after screening and identification of the multiple secondary transformants, secondary positive transformants were obtained. Based on the aforementioned secondary positive transformants, after induced expression and Chinese fermentation, the protein was purified to obtain recombinant XVII humanized collagen.

[0010] As some optional embodiments of this application, the linearization treatment refers to linearizing the recombinant humanized collagen protein particle or the recombinant lysine hydroxylase gene plasmid. The linearization enzyme digestion treatment conditions were 37℃ and 4h~6h; The enzyme digestion system is as follows: 10 μg~15 μg plasmid, 10 μL Quick Cut Sac I, 5 μL 10× Quick Cut buffer, and sterile water to make up to a total volume of 50 μL.

[0011] Add 0.1 volume of 3 M NaAc and 2.5 volume of anhydrous ethanol to the enzyme digestion system, and incubate overnight at -20°C. Centrifuge at 4°C and 13000 rpm for 20 min, and discard the supernatant. Add 700 μL of 75% ethanol to rinse, centrifuge at 13000 rpm for 20 min, discard the supernatant, and repeat once. Invert the EP tube on absorbent paper in a clean bench for about 10 min to remove as much water and residual ethanol as possible. Redissolve the plasmid with 20 μL of ddH2O, take 1 μL to dilute 10 times, and use one-drop to detect the nucleic acid concentration to obtain the linearized gene plasmid.

[0012] As some optional embodiments of this application, the electroconversion includes: Mix 80-100 μL of competent yeast cells with 8-10 μL of linearized plasmid, and transfer the mixture to a pre-chilled electroporation cuvette. Incubate on ice for 5-10 minutes. Select the yeast mode on the electroporator and perform electroporation. Add 1 mL of 1M sorbitol pre-chilled to 4°C to the electroporation cuvette, mix well, and transfer to a sterile EP tube. Incubate at 30°C for 1-2 hours. Spread 100-200 μL of the bacterial culture onto a solid plate, incubate at room temperature for 10 minutes, and then incubate upside down at 30°C for approximately 2-5 days until single colonies appear. The yeast competent cells include GS115 competent cells or recombinant engineered bacterial competent cells; the linearized plasmid includes the recombinant humanized collagen protein plasmid or the recombinant lysine hydroxylase gene plasmid. When the yeast competent cells are GS115 competent cells, the linearized plasmid is a recombinant humanized collagen protein plasmid, and the solid plate is a YPD plate containing 0.25 mg / mL Zeocin; When the yeast competent cells are recombinant engineered bacterial competent cells, the linearization plasmid is a recombinant lysine hydroxylase gene plasmid, and the solid plate is an MD solid plate.

[0013] On the other hand, this application also provides a recombinant XVII humanized collagen, prepared by the method described above.

[0014] As some optional embodiments of this application, the recombinant XVII humanized collagen has a molecular weight of 34897.46 Da and an isoelectric point of 10.80 for the key band of the protein component.

[0015] As some optional embodiments of this application, the recombinant XVII humanized collagen has a cell proliferation-promoting activity of 2230 U / mg, a cell adhesion-promoting activity of 645 U / mg, and a cell migration rate of 34%.

[0016] Compared with the prior art, the beneficial effects of this application are: This application optimizes and screens nucleotides from repetitive fragments in the human COL17 functional region. Using specific functional fragments, an amino acid sequence containing the integrin receptor site "RGD" (enhancing protein adhesion activity) and the skin follicle repair-related binding site "KGHK" was designed. This sequence was then co-expressed with the lysine hydroxylase (LH) gene in a Pichia pastoris system. The yeast expression system was optimized to improve the efficiency of lysine hydroxylation, thereby optimizing post-translational modifications of collagen proteins. Simultaneously, lysine hydroxylation increases the thermal stability and bioactivity of collagen, successfully yielding a recombinant humanized collagen protein with good bioactivity and stable safety. This recombinant humanized collagen protein can be stably produced industrially. It has lower production costs, more stable product quality, and good bioactivity, eliminating dependence on rare natural sources and meeting diverse research and clinical needs, thus showing broad application prospects in the biomedical field. Attached Figure Description

[0017] Figure 1 This is a pPICZαA-COL plasmid map related to the embodiments of this application; Figure 2 This is a pPIC3.5K-rhLH2 plasmid map related to the embodiments of this application; Figure 3 This is a PCR electrophoresis pattern of positive bacterial colonies involved in the embodiments of this application; Figure 4 The above are the SDS-PAGE results of the yeast engineered strain expressing recombinant humanized collagen involved in the embodiments of this application; Figure 5 This is a PCR electrophoresis image of positive bacterial colonies involved in the embodiments of this application; Figure 6 This is an SDS-PAGE image of the co-expressed protein in the yeast engineered strains involved in the embodiments of this application; Figure 7 The above are the SDS-PAGE results of the pilot-scale fermentation of the yeast engineered strain involved in the embodiments of this application; Figure 8 The results of SDS-PAGE analysis of purified supernatant protein involved in the embodiments of this application are shown. Figure 9 This is the deconvolution mass spectrum of recombinant humanized type XVII collagen involved in the embodiments of this application; Figure 10 This is an isoelectric point focused electrophoresis image related to an embodiment of this application; Figure 11 The infrared spectrum is shown in the embodiments of this application. Figure 12 This is a CD spectrum diagram related to an embodiment of this application; Figure 13The image shows the characteristics of a recombinant humanized collagen sample heated to 100°C for 30 minutes in the embodiments of this application; A represents the control group sample without heating; B represents the sample treated by heating to 100°C for 30 minutes. Figure 14 This is a graph showing the SDS-PAGE protein electrophoresis results of samples heated to 100°C according to the embodiments of this application; A represents the control group sample without heating; B represents the sample treated by heating at 100°C for 30 minutes. Figure 15 Analysis of the cell proliferation-promoting activity of recombinant humanized type XVII collagen involved in the embodiments of this application; Figure 16 Analysis of the cell adhesion-promoting activity of recombinant humanized type XVII collagen involved in the embodiments of this application; Figure 17 This application provides an analysis of the cell migration-promoting activity of recombinant humanized type XVII collagen involved in the embodiments of this application. Figure 18 The results of establishing a mouse skin lesion model after treatment with 8% sodium sulfide, as described in the embodiments of this application; A represents the macroscopic observation of the mouse back after treatment; B represents the observation under a CBS dermoscope. Figure 19 This is a comparison of skin repair and new hair growth in the hair removal area during the same time period for each group involved in the embodiments of this application. Detailed Implementation

[0018] It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0019] Type XVII collagen belongs to the collagen family. Unlike most collagens, human type 17 collagen (COL17 / COL17A1) is a transmembrane non-fibroblastic collagen, not an extracellular secretory collagen. Its structure encompasses extracellular collagen-like regions, transmembrane regions, and intracellular regions. It is a homogeneous trimer composed of three identical α1 (XVII) chains, which can be divided into three major structural regions: intracellular, transmembrane, and extracellular. Each α1 chain contains 1497 amino acids. Type XVII collagen is extremely rare in humans and animals, making its extraction extremely difficult. Due to its long amino acid chains and large molecular weight, both extraction and synthesis are very challenging, making mass production difficult. Therefore, similar raw materials and products are currently rare on the market.

[0020] With the development of synthetic biology technology, it has become possible to produce type XVII collagen using recombinant expression technology. Scientists can use recombinant DNA technology to express specific proteins in heterologous hosts (such as E. coli, yeast, insect cells, or mammalian cells). Recombinant type XVII collagen has the following advantages: First, it has higher purity and stability, can replace natural animal tissue collagen, and possesses good biocompatibility and controllable quality; Second, by obtaining recombinant type XVII humanized collagen through synthetic biology methods and combining it with specific enzymatic digestion processes, the transdermal absorption rate of the product can be improved, promoting the absorption of nutrients by the skin.

[0021] Human type 17 collagen, as a unique transmembrane collagen, plays an important role in maintaining skin homeostasis, skin repair, anti-aging, controlling hair loss and graying, and maintaining hair follicle stem cells. It shows broad application prospects in many fields such as medicine, beauty, and functional skin care products.

[0022] Type XVII collagen is an important component of various human tissues and is a transmembrane protein found in the skin, particularly abundant in the basement membrane zone at the junction of the epidermis and dermis. It plays a crucial role in maintaining skin structural integrity and promoting skin repair and regeneration. Furthermore, COL17A1 is also expressed in hair follicles, and its expression level is correlated with hair health, making it a potential target in skin care and regenerative medicine. The bioactivity of type XVII collagen depends on its specific three-dimensional structure and post-translational modifications, such as glycosylation. However, the natural scarcity of type XVII collagen and its complex structural characteristics pose significant challenges to its natural extraction, recombinant protein synthesis, purification, and large-scale production. Large-scale production of recombinant collagen requires efficient manufacturing processes and quality control measures. Current research attempts to produce type XVII collagen in various recombinant expression systems, including insect cells, mammalian cells, and bacterial systems. However, these systems have limitations in expression efficiency, proper protein modification, and feasibility for large-scale production, and face challenges in maintaining the protein's native structure, bioactivity, and in vitro environmental stability. Due to the aforementioned technical challenges, the clinical application and translation of COL17 faces significant bottlenecks. Although COL17 has shown potential therapeutic effects in various disease models, such as skin regeneration and wound healing, its clinical application is limited by the limited protein source and high production costs. This application uses its specific functional fragment as the basic repeating unit, performs predictive analysis on the amino acid sequence, optimizes the nucleotide sequence, and employs co-expression technology to produce a stable recombinant collagen product with good biological activity using a yeast expression system.

[0023] The Pichia pastoris expression system is a widely used eukaryotic expression system with the ability to modify proteins after translation (post-translational glycosylation), and is often used to produce recombinant proteins. However, the expression of recombinant proteins often faces problems such as low expression levels, improper protein folding, and poor stability. To address these issues, altering the nucleotide sequence of the gene to include more frequently used codons can improve protein translation efficiency. This helps reduce errors during translation, thereby increasing the proportion of correctly folded proteins.

[0024] In other words, human type XVII collagen plays an important role in maintaining skin homeostasis, skin repair, and maintaining the activity of hair follicle stem cells. However, type XVII collagen is naturally rare and has a complex structure. The lack of post-translational modification and other technical challenges in the recombinant expression production process leads to low yield, poor biological activity, and instability, which limits its translation into clinical applications.

[0025] To address the aforementioned issues and obtain stable, safe, bioactive, and widely applicable recombinant humanized collagen, this application optimized and screened nucleotides of the repetitive fragments in the human COL17 functional region. Based on specific functional fragments, an amino acid sequence containing the integrin receptor site "RGD" (enhancing protein adhesion activity) and the skin follicle repair-related binding site "KGHK" was designed. This sequence was co-expressed with the lysine hydroxylase (LH) gene in a Pichia pastoris system, optimizing the yeast expression system to improve the efficiency of lysine hydroxylation and optimize post-translational modifications of collagen proteins. Simultaneously, lysine hydroxylation enhances the thermal stability and bioactivity of collagen, successfully yielding a bioactive, stable, and safe recombinant humanized collagen. This recombinant humanized collagen can be stably produced industrially with lower production costs, more stable product quality, and good bioactivity. It eliminates dependence on rare natural sources, meets diverse research and clinical needs, and has broad application prospects in the biomedical field. The technical solution described in this application will be explained in detail below with reference to specific embodiments and experimental examples: Example 1: Construction, identification and preparation of recombinant humanized type XVII collagen engineered bacteria 1. Protein sequence selection The collagen peptide sequence described in this application is derived from human type XVII collagen (UniProt: NC_000010.11). Protein analysis was performed on the triple helix region of the mature collagen peptide, and the following amino acid sequences were selected from the peptide domain: C1 amino acid sequence (21aa, UniProt: KAI2557177.1, 18 to 38, SEQ ID No.1), C2 amino acid sequence (21aa, UniProt: KAI2557177.1, 21 to 41, SEQ ID No.2), and C3 amino acid sequence (12aa, UniProt: KAI2557177.1, 39 to 50, SEQ ID No.3). The C1 and C2 amino acid sequences were joined end-to-end to form the C4 collagen peptide (42aa, SEQ ID No.3), and the integrin receptor recognition site “RGD” was obtained from the C4 amino acid sequence. The C4 collagen peptide was linked end-to-end with the C3 amino acid sequence to form the C5 collagen peptide (54aa, SEQ ID No. 5). The skin follicle repair recognition site "KGHK" was obtained from the amino acid sequence of the C5 collagen peptide. Based on previous research on recombinant collagen, the basic unit sequence (SEQ ID No. 5) was selected for expression with 7 tandem repeats (SEQ ID No. 6), and the predicted molecular weight was approximately 36.2 kDa.

[0026] SEQ ID No.1(UniProt:KAI2557177.1,18 to 38): GPKGDRGPAGPPGHPGPPGPR SEQ ID No.2(UniProt:KAI2557177.1,18 to 38): GDRGPAGPPGHPGPPGPRGHK SEQ ID No.3(UniProt:KAI2557177.1,18 to 38): GHKGEKGDKGDQ SEQ ID No. 4: GPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHK SEQ ID No. 5: GPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQ SEQ ID No.6: GPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQGPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQGPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQGPKGDRGPAGPPGHPGPPGPRGDRGPA GPPGHPGPPGPRGHKGHKGEKGDKGDQGPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQGPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQGPKGDRGPAGPPGHPGPPGPRGDRGPAGPPGHPGPPGPRGHKGHKGEKGDKGDQ A terminator was added to the C-terminus, and the DNA sequence encoding the protein was optimized for Pichia pastoris preference (SEQ ID No. 7) to make the recombinant protein more suitable for expression in Pichia pastoris.

[0027] SEQ ID No. 7: The optimized base sequence of recombinant humanized collagen (SEQ ID No. 7) at the 5' end, i.e. Xho I. The base sequence AAAAGA was added between the target gene sequence and the gene fragment, and the gene fragment was synthesized by Genscript Biotech Co., Ltd. The synthesized gene fragment was then processed through... EcoR I and Not The restriction enzyme site I was inserted into the pPICZαA plasmid to obtain recombinant humanized collagen protein plasmids. Figure 1 ).

[0028] The amino acid sequence of the lysine hydroxylase described in this application is the human gene LH2 (UniProt: NC_000003.12), and its specific amino acid sequence is (SEQ ID No. 8), with a predicted molecular weight of approximately 77.2 kDa.

[0029] SEQ ID No. 8: MKEVMEHYADQDDLVVMFTECFDVIFAGGPEEVLKKFQKANHKVVFAADGILWPDKRLADKYPVVHIGKRYLNSGGFIGYAPYVNRIVQQWNLQDNDDDQLFYTKVYIDPLKREAAINITLDHKCKIFQTLNGAVDEVVLKFENGKARAKNTFYETLPVAINGNGPT KILLNYFGNYVPNSWTQDNGCTLCEFDTVDLSAVDVHPNVSIGVFIEQPTPFLPRFLDILLTLDYPKEALKLFIHNKEVYHEKDIKVFFDKAKHEIKTIKIVGPEENLSQAEARNMGMDFCRQDEKCDYYFSVDADVVLTNPRTLKILIEQNRKIIAPLVTRHGKLW SNFWGALSPDGYYARSEDYVDIVQGNRVGVWNVPYMANVYLIKGKTLRSEMNERNYFVRDKLDPDMALCRNAREMTLQREKDSPTPETFQmLSPPKGVFMYISNRHEFGRLLSTANYNTSHYNNDLWQIFENPVDWKEKYINRDYSKIFTENIVEQPCPDVFWFPI FSEKACDELVEEMEHYGKWSGGKHHDSRISGGYENVPTDDIHMKQVDLENVWLHFIREFIAPVTLKVFAGYYTKGFALLNFVVKYSPERQRSLRPHHDASTFTINIALNNVGEDFQGGGCKFLRYNCSIESPRKGWSFMHPGRLTHLHEGLPVKNGTRYIAVSFIDP A 6×His terminator was added to the C-terminus. The lysine hydroxylase gene sequence was optimized (SEQ ID No. 9) to better suit its expression in Pichia pastoris.

[0030] SEQ ID No. 9: CATCATCATCATCATCAT The optimized DNA sequence of lysine hydroxylase (LH2) (SEQ ID No. 9) was commissioned to GenScript Biotech Inc. for gene fragment synthesis. The synthesized gene fragment was then processed through... Eco RⅠ and Not The restriction enzyme site I was inserted into the pPIC3.5K plasmid to obtain the recombinant lysine hydroxylase gene plasmid. Figure 2 ).

[0031] 2. Construction and screening of recombinant engineered bacteria 2.1 Obtaining recombinant plasmids The optimized base sequences (SEQ ID No. 7, SEQ ID No. 9) were commissioned to GenScript Biotech Co., Ltd. for gene fragment synthesis. After sequencing verification, the corresponding plasmids and stab bacteria were provided. High-concentration plasmids were extracted after large-scale culture for later use.

[0032] 2.2 Linearization of pPICZαA-COL plasmid Using QuickCut Sac I. Linearization and enzyme digestion were performed on 20 μg of plasmid pPICZαA-COL extracted in 2.1. The digestion conditions were 37℃ for 5 h. The digestion system is as follows: Plasmid: 10μg~15μg; Quick Cut Sac I: 10 μL; 10× Quick Cut buffer: 5 μL; Total volume: 50 μL.

[0033] After electrophoresis verification, add 0.1 volume of 3 M NaAc (pH 5.2) and 2.5 volume of anhydrous ethanol, and incubate overnight at -20°C. Centrifuge at 13000 rpm for 20 min at 4°C, and discard the supernatant; add 700 μL of 75% ethanol to rinse, centrifuge at 13000 rpm for 20 min, discard the supernatant, and repeat once; invert the EP tube on absorbent paper in a clean bench for about 10 min to remove as much water and residual ethanol as possible, redissolve the plasmid with 20 μL of ddH2O, take 1 μL to dilute 10-fold, and use one-drop to detect the nucleic acid concentration.

[0034] 2.2 Preparation of GS115 competent cells After streaking GS115 onto a plate, a single colony was picked and inoculated into 20 mL of YPD medium and incubated at 30°C and 225 rpm for 24 h. Then, it was transferred to 50 mL of YPD liquid medium at a 1:1000 inoculation ratio and incubated at 30°C and 225 rpm until OD (dose elapsed). 6001.3-1.5; Transfer the bacterial culture to a sterile 50 mL centrifuge tube, centrifuge at 3000 rpm for 5 min at 4 °C, discard the supernatant and collect the bacterial cells; resuspend the bacterial cell pellet in 50 mL of pre-chilled sterile ultrapure water, centrifuge at 3000 rpm for 5 min at 4 °C, discard the supernatant and resuspend the bacterial cell pellet in 50 mL of pre-chilled sterile ultrapure water; centrifuge at 3000 rpm for 5 min at 4 °C, discard the supernatant and resuspend the cell pellet in 40 mL of pre-chilled sterile 1 M sorbitol; centrifuge at 3000 rpm for 5 min at 4 °C, discard the supernatant and resuspend the cell pellet in 100 μL-150 μL of pre-chilled sterile 1 M sorbitol, gently vortex to mix, place on ice, and set aside for use.

[0035] 2.3 Electroporation into GS115 competent cells Mix 100 μL of competent yeast cells with 10 μL of linearized plasmid and transfer to a pre-chilled electroporation cuvette. Immediately incubate on ice for 5 min. Select the yeast mode on the electroporator and perform electroporation. Then, immediately add 1 mL of pre-chilled 1M sorbitol to the cuvette, mix thoroughly, and transfer the mixture to a sterile EP tube. Incubate at 30°C for 1-2 h. Spread 100 μL-200 μL of the bacterial culture onto a YPD plate containing 0.25 mg / mL Zeocin. Incubate at room temperature for 10 min, then incubate upside down at 30°C for approximately 2-5 days until single colonies appear.

[0036] 2.4 Screening of positive transformants Single colonies were picked from YPD plates and transferred to 96-well plates containing 200 μL of YPD medium (containing 0.5 mg / mL Zeocin), and cultured at 30°C. After 48 h, the bacterial culture was homogenized, and 10 μL was transferred from each well to a new 96-well plate (containing 190 μL of YPD). The culture was then repeated for 24 h. After 24 h, the bacterial culture in a third 96-well plate was homogenized, and 1 μL was spotted onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin, respectively, and cultured. If the transformant could grow on plates containing a high concentration of 3 mg / mL Zeocin, it indicated that the transformant contained multiple copies of the target gene. This screening step yielded a yeast strain capable of efficiently expressing recombinant humanized collagen.

[0037] 2.5 Identification of positive transformants Take 50 μL of the remaining bacterial culture 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 the freeze-thaw cycle, centrifuge at 12000 rpm for 5 min, take the supernatant as a PCR template, and identify whether the target gene has been integrated into the yeast chromosome by PCR amplification.

[0038] Upstream primer: 5'AOX1(5'-GACTGGTTCCAATTGACAAGC-3') Downstream primer: 3'AOX1(5'-GCAAATGGCATTCTGACATCC-3') PCR conditions: 98℃ pre-denaturation for 5 min, 98℃ heat denaturation for 50 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ annealing for 10 min.

[0039] The amplified products were subjected to 1.0% agarose gel electrophoresis to determine whether the expected gene fragment (approximately 1683 bp) had been amplified. Figure 3 ).

[0040] 2.6 Expression of recombinant humanized collagen The culture medium formula used is as follows: 1) YPD complete medium (solid medium containing 2% agar): Yeast extract 10g / L, peptone 20g / L, glucose 20g / L; 2) MD solid medium (solid medium containing 2% agar): YNB contains 13.4 g / L amino acid-free nitrogen source; 0.4 mg / L biotin; 20 g / L glucose; 3) BMGY yeast growth medium: Yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, K2HPO4 11.8g / L, add water to 890mL, sterilize at 121℃ for 20 minutes, then after the temperature drops to 60℃, add 10×YNB 100mL (13.4g / L) and 500×Biotin 1mL (4×10) on a clean bench. -4 (g / L), 10 mL of glycerol.

[0041] 4) BMMY yeast induction medium: Yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, K2HPO4 11.8g / L, add water to 895mL, sterilize at 121℃ for 20 minutes, then after the temperature drops to 60℃, add 100mL of 100×YNB (13.4g / L) and 1mL of 500×Biotin (4×10) on a clean bench. -4 (g / L), 5 mL of methanol.

[0042] The specific procedure is as follows: Select 5 single colonies and inoculate them into BMGY medium, incubate at 30℃ and 220 rpm for 24 hours until OD reaches the target value. 600nmThe initial OD value was 2.0–6.0. ​​The volume of the BMGY culture medium was adjusted based on the measured OD value. The cells were collected at 3000 rpm for 10 min and resuspended in an equal volume of BMMY medium to achieve an initial OD value of 2.0. The culture was continued at 30℃ and 220 rpm, with 0.5% methanol added to the medium every 24 h. After induction, the bacterial culture sample was collected 72 h later, and the expression supernatant was collected by centrifugation. The molecular weight (approximately 36 kDa) and expression level of the target protein were analyzed by SDS-PAGE electrophoresis. Figure 4 ).

[0043] 2.7 Secondary electroporation of recombinant humanized collagen engineered strains use Sal Ⅰ. After linearizing and digesting the pPIC3.5K-LH2 plasmid with enzymes (refer to step 2.1), electroporate it into competent recombinant engineered bacterial cells prepared from the high-copy strain screened in step 2.5 (refer to step 2.2). Spread 100 μL to 200 μL of the bacterial culture onto MD agar plates, incubate at room temperature for 10 min, and then incubate upside down at 30°C for approximately 2 to 5 days until single colonies appear.

[0044] 2.8 Screening of positive transformants Add 2 mL of sterile double-distilled water to the surface of an MD plate, and then gently scrape off the His from the plate surface using a sterile triangular applicator. + Transformants were collected and transferred to 50 mL centrifuge tubes. OD counts were performed using a spectrophotometer. 600 (≈5×10⁷ cells / mL). Dilute with sterile water to a concentration of 10⁵ cells / mL, spread on YPD plates containing 0.5 mg / mL G418, invert, and incubate at 30°C for 3-4 days until single colonies appear. Pick a single colony from the YPD plate and transfer it to a 96-well plate containing 200 μL of YPD medium, and continue incubation at 30°C. After 48 hours, rehydrate the bacterial suspension, and transfer 10 μL from each well to a new 96-well plate (containing 190 μL of YPD), and continue incubation for 24 hours, repeating the above steps once. After 24 hours, rehydrate the bacterial suspension in a third 96-well plate, and spot 1 μL onto YPD plates containing 1 mg / mL, 2 mg / mL, and 4 mg / mL G418 respectively, and continue incubation. If the transformant can grow on a plate containing a high concentration of 4 mg / mL G418, it indicates that the transformant contains multiple copies of the target gene. This screening step yields recombinant humanized collagen-producing yeast strains that can express collagen efficiently.

[0045] 2.8 Identification of positive strains Pick a small amount of single colony from step 2.7 above and dissolve it in 10μL-20μL of sterile water. Boil in a water bath for 10 min, freeze in liquid nitrogen for 30 min, boil in a water bath for 10 min, repeat the freeze-thaw cycle, centrifuge at 12000 rpm for 5 min, and use the supernatant as a PCR template. Use PCR amplification to identify whether the target gene has been integrated into the yeast chromosome.

[0046] P4H-F: 5'-TTCGAAGGATCCTACGTAGAAT- 3' P4H-R: 5' -CCGCTCAATGATGATGATGATG- 3' PCR conditions: 98℃ pre-denaturation for 5 min, 98℃ heat denaturation for 50 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 72℃ annealing for 10 min.

[0047] The amplified products were subjected to 1.0% agarose gel electrophoresis to determine whether the expected gene fragment (approximately 2231 bp) had been amplified. Figure 5 ).

[0048] 3. Induced expression of recombinant humanized collagen in yeast engineered strains The culture medium formula used is as follows: 1) YPD complete medium (solid medium containing 2% agar): Yeast extract 10g / L, peptone 20g / L, glucose 20g / L; 2) MD solid medium (solid medium containing 2% agar): YNB contains 13.4 g / L amino acid-free nitrogen source; 0.4 mg / L biotin; 20 g / L glucose; 3) BMGY yeast growth medium: Yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, K2HPO4 11.8g / L, add water to 890mL, sterilize at 121℃ for 20 minutes, then after the temperature drops to 60℃, add 10×YNB 100mL (13.4g / L) and 500×Biotin 1mL (4×10) on a clean bench. -4 (g / L), 10 mL of glycerol.

[0049] 4) BMMY yeast induction medium: Yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, K2HPO4 11.8g / L, add water to 895mL, sterilize at 121℃ for 20 minutes, then after the temperature drops to 60℃, add 100mL of 100×YNB (13.4g / L) and 1mL of 500×Biotin (4×10) on a clean bench.-4 (g / L), 5 mL of methanol.

[0050] The specific procedure is as follows: Select a single colony and inoculate it onto BMGY medium, incubate at 30℃ and 220 rpm for 24 hours until OD reaches the target level. 600 =2-6. Adjust the volume of BMGY culture medium according to the measured OD value, collect the cells at 3000 rpm for 10 min, and resuspend the cells in an equal volume of BMMY medium to achieve an initial OD value of 2.0. Continue culturing at 30℃ and 220 rpm, adding 0.5% methanol, 1% trace elements (PTM1), and ascorbic acid (final concentration 10 mM) to the medium every 24 h. Collect bacterial samples at 24 h, 48 h, and 72 h after induction, lyse the cells, and collect the expression supernatant by centrifugation. Analyze the molecular weight (approximately 77 kDa) and expression level of the target protein by SDS-PAGE electrophoresis. Figure 6 The specific procedure is as follows: Take 40 μL of supernatant, add 10 μL of 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ water for 10 min, add 10 μL to each well of SDS-PAGE protein gel, treat at 80V for 2 h, stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 40 min, and then decolorize with protein destaining solution (10% acetic acid, 5% ethanol) to obtain GS115 / pPICZαA-COL / P4H Pichia pastoris engineered strain.

[0051] 4. Chinese-style fermentation using engineered yeast Seed culture medium YPD (formulation: yeast extract 10g / L, peptone 20g / L, glucose 20g / L); Fermentation medium: 85% H3PO4 26.7 mL / L; CaSO4·2H2O 1.175 g / L; K2SO4 18.2 g / L; MgSO4·7H2O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM 14.35 mL / L; Feed culture medium (50% w / v glycerol, with 12 mL PTM1 trace element per liter); Induction medium (100% methanol, with 12 mL of PTM1 trace element added per liter); PTM1 trace element was added after the fermentation medium was sterilized at high temperature and cooled to room temperature, and the pH was adjusted to 5.0 with ammonia water. The PTM1 solution was prepared according to the Invitrogen manual, with the following specific formulation: 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 and stored at 4 °C.

[0052] 2L of freshly prepared fermentation medium (Bailun Biotechnology Co., Ltd.) was added to a 5L fermenter, and the fermenter was sterilized in a steam sterilizer at 121°C for 15 minutes. After the fermenter cooled, the electrodes were calibrated, and inoculation was initiated. During the fermenter preparation stage, engineered Pichia pastoris strains (GS115 / pPICZαA-COL / P4H) stored in an ultra-low temperature freezer were inoculated into a medium containing 50 mL of liquid YPD and cultured overnight at 30°C and 220 rpm in a constant temperature shaker to activate the cells. The primary seed culture was then transferred at a 5% (v / v) inoculation volume to a medium containing 200 mL of YPD for scale-up culture until the cell OD reached 100%. 600 From step 4-6, secondary seed culture was obtained. All of the secondary seed culture was added to the fermenter (10% inoculum, v / v), along with 8.7 mL of PTM1 micronutrients, for batch fermentation. The temperature for the glycerol growth and glycerol feeding phases was set at 30 °C, and the temperature for the methanol induction phase was set at 25 °C. Dissolved oxygen (DO) in the fermenter was maintained between 20% and 50% by controlling the stirring speed (500-600 rpm), aeration rate (2-6 L / min), and feed rate. 50% (w / w) ammonia was automatically added to maintain the culture medium pH at approximately 5.0. Antifoaming agent was added to remove foam during fermentation. During the glycerol culture phase, a continuous increase in DO indicated that the glycerol in the culture medium had been depleted by the cells. At this point, approximately 400 mL of 50% (w / v) glycerol was added. Once the cell wet weight reached 220 g / L, glycerol addition was stopped, and the cells were starved for approximately 0.5 h. Once the dissolved oxygen (DO) in the tank reached its maximum again, methanol was added to induce the production of recombinant humanized collagen. The feeding rate was periodically adjusted to maintain DO levels within a cyclical fluctuation range of 20%-50%. Bacterial culture samples were collected at 24 h, 48 h, and 72 h after induction, and the expression supernatant was collected by centrifugation. The molecular weight (approximately 36 kDa) and expression level of the target protein were analyzed by SDS-PAGE electrophoresis. Figure 7 ).

[0053] 5. Purification The supernatant of the culture medium was collected by centrifugation, and the fermentation supernatant and bacterial cells were separated using a solid-liquid separation system. The column was equilibrated with phosphate buffer (20 mM NaH2PO4, pH 5.0) until the conductivity and A280 absorbance remained constant. The sample loading flow rate was set to 20 cm / h, and the UV A280 absorbance was monitored. Sample loading began when the absorbance increased. After loading, the cation exchange medium was equilibrated again with phosphate buffer until the UV absorbance and conductivity reached their minimum and remained unchanged. The protein was then eluted with a buffer containing NaH2PO4-NaCl (0.5 M) and collected. After ultrafiltration, buffer replacement, and concentration, the recombinant humanized collagen stock solution was obtained. The molecular weight and purity were determined using SDS-PAGE. Figure 8 ).

[0054] Example 2: Identification of Recombinant Collagen 1. Molecular weight analysis: The amino acid sequence of the recombinant humanized collagen was predicted by Expasy, with a theoretical molecular weight of approximately 36.2 kDa. However, the SDS-PAGE electrophoresis results showed a molecular weight higher than the theoretical value, which may be due to glycosylation modification (high-mannose N-glycans) in the Pichia pastoris expression system. The protein sample, after EndoH restriction enzyme digestion, was analyzed by the Public Technology Center of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and the results are as follows: Figure 9 The detected molecular weight of recombinant humanized XVII collagen was 34897.46 Da, which is basically consistent with the theoretical molecular weight of 36248.64 Da.

[0055] 2. Protein isoelectric point analysis Specific implementation methods: Sample preparation: Replace 80 μg of each sample solution with 8M urea in the buffer, and add an appropriate amount to 5 μL of amphoteric electrolyte before proceeding with subsequent experiments. Plate isoelectric focusing electrophoresis: Prepare a plate isoelectric focusing electrophoresis gel, and spot an appropriate amount of sample onto a filter paper strip for isoelectric focusing electrophoresis. After focusing, fix and stain the gel, then thoroughly destain and scan. Isoelectric point analysis: Perform isoelectric point analysis using ImageMaster 5.0 software.

[0056] Figure 10 The left lane is the marker, and the right lane contains recombinant humanized collagen (15 μg sample; the numbers on the left indicate the isoelectric points of the major bands in the marker, and the numbers on the sample indicate the band numbers). The calculated isoelectric point of the major bands in the protein component of the sample is 10.80.

[0057] Example 3 Characterization and analysis of recombinant collagen 1. Fourier transform infrared spectroscopy (FT) IR analysis: Infrared spectroscopy is an effective method for studying the structure of biological macromolecules and the conformation of polypeptide chains. The characteristic absorption peaks of collagen groups can be detected by infrared spectroscopy. The Shanghai Institute of Materia Medica, Chinese Academy of Sciences, was commissioned to conduct the analysis. The recombinant humanized collagen was purified, lyophilized, ground into powder using KBr, and then compressed into tablets. The samples were then scanned and detected under room temperature and low humidity conditions. Figure 11 Infrared spectral scanning of collagen purified samples. Figure 11 It can be seen that the wavenumbers of its amide A, amide I, amide II, and amide III all conform to the structural characteristics of recombinant collagen.

[0058] 2. Circular dichroism (CD) spectral analysis: According to the standard "YY / T 1888-2023 Recombinant Humanized Collagen", when applicable, the CD spectrum of recombinant humanized collagen has a negative peak at a wavelength near 198 nm. We used CDNN software to analyze the secondary structure of the sample ( Figure 12 According to the fitting calculation, the helix accounts for 66.83%.

[0059] Example 4: High-Temperature Denaturation Analysis of Recombinant Protein Specific experimental method: Heat the water bath to 100℃, remove the sample to be tested (the sample is placed in a sealed vial) from the 4℃ refrigerator, place it in boiling water and heat for 30 minutes, then remove the sample and observe the appearance changes (recombinant humanized type XVII collagen remains a clear and transparent liquid). Figure 13 Sampling and SDS-PAGE electrophoresis were performed (the protein showed minimal degradation after boiling in hot water). Figure 14 ).

[0060] Example 5: In vitro cellular activity analysis of recombinant humanized type XVII collagen 1. Cell proliferation-promoting activity According to the standard "YY / T 1888-2023 Recombinant Humanized Collagen," the dose-response relationship of recombinant collagen coating gradient concentrations was investigated using cell culture plates. The specific detection method was the MTT assay (performed according to the kit instructions), with cell culture plates without recombinant collagen coating used as a control to analyze cell proliferation rate. The results showed that the cell proliferation-promoting activity of recombinant humanized type XVII collagen was 2230 U / mg (…). Figure 15 ).

[0061] 2. Cell adhesion promotion activity According to the standard "YY / T 1888-2023 Recombinant Humanized Collagen", cell adhesion can be easily evaluated through a cell adhesion rate assay. The specific experimental method is as follows: Recombinant human COL17 is pre-diluted to 0.5 μg / mL with PBS. After pre-dilution, it is serially diluted 2-fold in 96-well plates, resulting in 10 dilutions. 50 μL of different dilutions of FN sample is added to each well. A negative control (without recombinant humanized collagen) is included, with 50 μL of PBS added as a control. The plates are incubated overnight at 4°C. After incubation, the liquid in the plates is discarded, and 100 μL of 30 g / L BSA is added to each well for blocking. The plates are then incubated at 37°C for 1 hour. The liquid in the plates is then discarded, and MDBK cell suspension (resuspended in serum-free medium) is added. The cell seeding density is 1.0 × 10⁶ cells / well. 5 Cells were seeded at a concentration of 100 μL / mL, and incubated for 5 h. After incubation, the cell plates were washed three times with PBS, and cell adhesion was observed under a microscope. Five points (excluding the edge) were selected under 200x magnification to count adherent cells. The median effective concentration (ED50) of recombinant human COL17 was determined based on the count results and a fitted curve. 50 The potency was calculated. The results showed that the cell adhesion-promoting activity of recombinant humanized type XVII collagen was 645 U / mg. Figure 16 ).

[0062] 3. Promotes cell migration or transition activity According to the standard "YY / T 1888-2023 Recombinant Humanized Collagen," recombinant collagen used for tissue repair and reconstruction should facilitate cell recognition and migration. The specific detection method uses a cell scratch assay, with cell culture medium without recombinant collagen as a control. The cell migration rate is calculated by comparing the number of cells migrating from the same scratch site at different time points. The results showed that recombinant humanized type XVII collagen promoted cell migration by 34% (…). Figure 17 ).

[0063] Example 6: Efficacy analysis of recombinant humanized type XVII collagen in skin and hair follicle repair and hair regeneration. 1. Analysis of the efficacy of hair follicle repair The testing was commissioned to the laboratory of the School of Pharmacy, Anhui Medical University. Specific experimental methods: Hair follicles in the anagen phase were isolated from healthy adult female black C57 mice, and the portion above the sebaceous gland opening was removed. The follicles were then cultured in Williams E medium. Mouse hair follicle culture was performed in 24-well plates, with one follicle per well. Each well contained 1 mL of Williams E medium (2.0 mmol / L L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin) and was incubated at 37°C in a 5% CO2 incubator. The experiment was divided into two groups, with 10 anagen-phase follicles in each group. The control group received sterile saline in Williams E medium; the experimental group received 0.5 g / L recombinant human XVII collagen in Williams E medium. Hair growth was observed on days 1, 3, and 5 of culture using an inverted microscope with a micrometer on the eyepiece. The morphological changes of the hair bulb were also observed under the inverted microscope. Statistical analysis was performed on the experimental data, with statistical variables expressed as ˉx±s. A two-sample t-test was used, with a two-sided α=0.05. The statistical results of the experimental data are shown in Table 1. On day 5 of hair growth culture, the average hair growth length of the control group was (0.287±0.04) mm, while that of the experimental group was (0.494±0.05) mm. The hair growth length of the control group was significantly lower than that of the experimental group (P<0.001).

[0064] Table 1. Hair growth in each group at different time points

[0065] 2. Analysis of Hair Regeneration Efficacy The testing was commissioned to the Laboratory of the School of Pharmacy, Anhui Medical University. Specific experimental methods: Twelve healthy adult black female C57 mice were selected and housed in a clean-grade environment, provided with ample food and water, and fed and watered daily. The animals' physiological activities were monitored. They were randomly divided into two groups of six mice each. The back hair of the mice was shaved. All shaved mice were treated with 8% sodium sulfide on their backs for hair removal. After 5 minutes, the sodium sulfide residue was washed away with purified water, artificially creating a mouse skin damage model. Subcutaneous observation was performed using a CBS dermoscope after treatment. Figure 18 ).from Figure 18The observation results showed that the model caused damage to the skin around the hair follicles, but hair follicles were still visible underneath. Mice in the experimental group received a subcutaneous injection of 0.2 mL of a 1.0 mg / L recombinant human type XVII collagen solution in the rib area, while control mice received a subcutaneous injection of 0.2 mL of sterile PBS in the rib area. Injections were given every 2 days for a total of 7 injections. After the skin lesion model was established, the mice's skin repair status, hair growth status, and skin color changes were observed daily. Photos were taken every 5 days, and skin changes and hair growth time were recorded until all hair appeared. Typical changes in the experimental and control groups were compared. Figure 19 ).from Figure 19 The results showed that the experimental group had faster skin repair and hair growth. By day 10, hair growth had appeared in some areas of the lesion and the scar area had shrunk. By day 15, almost all the lesion areas had hair growth, and the hair removal area had reached 80% hair growth. No scar areas showing signs of healing were visible. In contrast, the control group mainly showed hair regrowth around the hair removal area, and only a small amount of hair regrowth in the lesion area. In summary, this application utilizes specific functional fragments from the functional region of recombinant humanized type XVII collagen for amino acid sequence design. This results in the recombinant humanized type XVII collagen prepared in this application containing integrin receptor sites that enhance protein adhesion activity and skin follicle repair-related binding sites. Furthermore, it co-expresses these sites with the lysine hydroxylase (LH) gene in the Pichia pastoris system, solving the post-translational modification problem of humanized type XVII collagen in recombinant expression production. This increases the thermal stability and biological activity of the recombinant humanized type XVII collagen. The recombinant humanized type XVII collagen prepared in this application exhibits good stability even after heating at 100℃ for 30 minutes, and is not easily denatured or degraded by high temperatures. This recombinant humanized type XVII collagen can be stably produced industrially, with lower production costs, more stable product quality, and good biological activity. It eliminates dependence on rare natural sources, meets diverse research and clinical needs, and shows broad application prospects in multiple fields such as medicine, beauty, and functional skincare products.

[0066] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing recombinant XVII humanized collagen, characterized in that, Includes the following steps: Obtain the amino acid sequence containing the integrin receptor site "RGD" and the binding site "KGHK"; Based on the amino acid sequence, recombinant humanized collagen protein particles were obtained; Based on the human gene LH2, a recombinant lysine hydroxylase gene plasmid was obtained. Recombinant XVII humanized collagen was obtained based on the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid.

2. The method for preparing recombinant XVII humanized collagen according to claim 1, characterized in that, The process of obtaining the amino acid sequence containing the integrin receptor site "RGD" and the binding site "KGHK" includes: Protein analysis was performed on the triple helix region of the mature peptide derived from human type XVII collagen. The C1 amino acid sequence shown in SEQ ID No. 1, the C2 amino acid sequence shown in SEQ ID No. 2, and the C3 amino acid sequence shown in SEQ ID No. 3 of the human type XVII collagen peptide domain fragment were selected. The C1 amino acid sequence and the C2 amino acid sequence were connected end to end to form the C4 collagen peptide shown in SEQ ID No.

4. The integrin receptor site "RGD" was obtained from the C4 amino acid sequence. The C4 collagen peptide is linked end-to-end with the C3 amino acid sequence to form the C5 collagen peptide as shown in SEQ ID No. 5, and the skin hair follicle repair binding site "KGHK" is obtained from the C5 collagen peptide.

3. The method for preparing recombinant XVII humanized collagen according to claim 2, characterized in that, The process of obtaining recombinant humanized collagen protein particles based on the amino acid sequence includes: After repeating the C5 collagen peptide tandemly 7 times, the sequence shown in SEQ ID No. 6 was obtained. A terminator was added to its C-terminus, and the DNA sequence encoding the protein was optimized by Pichia pastoris preference design to obtain the optimized base sequence shown in SEQ ID No.

7. After adding the base sequence AAAAGA to the 5' end of the optimized base sequence, gene fragment synthesis was performed. The synthesized gene fragment was then processed... EcoR I and Not The restriction enzyme site I was inserted into the pPICZαA plasmid to obtain recombinant humanized collagen protein plasmid.

4. The method for preparing recombinant XVII humanized collagen according to claim 1, characterized in that, The recombinant lysine hydroxylase gene plasmid obtained based on the human LH2 gene includes: The human gene LH2 with an amino acid sequence such as SEQ ID No. 8 was optimized by adding 6×His and a terminator to the C-terminus, resulting in the optimized DNA sequence such as SEQ ID No.

9. The optimized DNA sequence was used to synthesize gene fragments, and the fragments were inserted into the pPIC3.5K plasmid through the EcoRI and NotRI restriction sites to obtain the recombinant lysine hydroxylase gene plasmid.

5. The method for preparing recombinant XVII humanized collagen according to claim 1, characterized in that, The recombinant XVII humanized collagen protein obtained based on the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid includes: Gene fragments were synthesized from the recombinant humanized collagen protein plasmid and the recombinant lysine hydroxylase gene plasmid to obtain pPICZαA-COL plasmid and pPIC3.5K-rhLH2 plasmid, respectively. After linearizing the pPICZαA-COL plasmid, it was electroporated into GS115 competent cells to obtain multiple transformants; after screening and identification of the multiple transformants, positive transformants were obtained; based on the positive transformants, recombinant engineered bacterial competent cells were prepared. After linearizing the pPIC3.5K-rhLH2 plasmid, it was electroporated into the competent cells of the recombinant engineered bacteria to obtain multiple secondary transformants; after screening and identification of the multiple secondary transformants, secondary positive transformants were obtained. Based on the aforementioned secondary positive transformants, after induced expression and Chinese fermentation, the protein was purified to obtain recombinant XVII humanized collagen.

6. The method for preparing recombinant XVII humanized collagen according to claim 5, characterized in that, The linearization treatment refers to linearizing the recombinant humanized collagen protein particle or the recombinant lysine hydroxylase gene plasmid with enzyme digestion. The linearization enzyme digestion treatment conditions were 37℃ and 4h~6h; The enzyme digestion system is as follows: 10 μg~15 μg plasmid, 10 μL Quick Cut Sac I, 5 μL 10× Quick Cut buffer, and sterile water to make up to a total volume of 50 μL; Add 0.1 volume of 3 M NaAc and 2.5 volume of anhydrous ethanol to the enzyme digestion system, and incubate overnight at -20°C. Centrifuge at 4°C and 13000 rpm for 20 min, and discard the supernatant. Add 700 μL of 75% ethanol to rinse, centrifuge at 13000 rpm for 20 min, discard the supernatant, and repeat once. Invert the EP tube on absorbent paper in a clean bench for about 10 min to remove as much water and residual ethanol as possible. Redissolve the plasmid with 20 μL of ddH2O, take 1 μL to dilute 10 times, and use one-drop to detect the nucleic acid concentration to obtain the linearized gene plasmid.

7. The method for preparing recombinant XVII humanized collagen according to claim 5, characterized in that, The electroconversion includes: Mix 80-100 μL of competent yeast cells with 8-10 μL of linearized plasmid, and transfer the mixture to a pre-chilled electroporation cuvette. Incubate on ice for 5-10 minutes. Select the yeast mode on the electroporator and perform electroporation. Add 1 mL of 1 M sorbitol pre-chilled to 4°C to the electroporation cuvette, mix well, and transfer to a sterile EP tube. Incubate at 30°C for 1-2 hours. Spread 100-200 μL of the bacterial culture onto a solid plate, incubate at room temperature for 10 minutes, and then incubate upside down at 30°C for approximately 2-5 days until single colonies appear. The yeast competent cells include GS115 competent cells or recombinant engineered bacterial competent cells; the linearized plasmid includes the recombinant humanized collagen protein plasmid or the recombinant lysine hydroxylase gene plasmid. When the yeast competent cells are GS115 competent cells, the linearized plasmid is a recombinant humanized collagen protein plasmid, and the solid plate is a YPD plate containing 0.25 mg / mL Zeocin; When the yeast competent cells are recombinant engineered bacterial competent cells, the linearization plasmid is a recombinant lysine hydroxylase gene plasmid, and the solid plate is an MD solid plate.

8. A recombinant XVII humanized collagen, characterized in that, Prepared by the method described in any one of claims 1-8.

9. The recombinant XVII humanized collagen according to claim 8, characterized in that, The recombinant XVII humanized collagen has a molecular weight of 34897.46 Da and an isoelectric point of 10.80 for the key band of the protein component.

10. The recombinant XVII humanized collagen according to claim 8, characterized in that, The recombinant XVII humanized collagen exhibited a cell proliferation-promoting activity of 2230 U / mg, a cell adhesion-promoting activity of 645 U / mg, and a cell migration rate of 34%.

Images