A Highly Active Recombinant Type XVII Collagen and Its Rapid Preparation Method and Application

Abstract

This invention relates to a highly active recombinant type XVII collagen and its rapid preparation method and applications, belonging to the field of bioengineering technology. The amino acid sequence of the highly active recombinant type XVII collagen is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. This invention also provides a method for preparing and applying this recombinant type XVII collagen. The highly active recombinant type XVII collagen of this invention exhibits good biological activity, possessing excellent cell adhesion, cell proliferation, and cell migration promotion effects. It can be compounded with other proteins to become raw materials for tissue engineering products, cosmetics, health products, or pharmaceuticals. Furthermore, by integrating the highly active recombinant type XVII collagen gene into different locations in the yeast genome using three different vectors, the copy number of the target gene is increased, thereby significantly increasing the expression level of recombinant type XVII collagen and effectively improving the yield of highly active recombinant type XVII collagen.

Description

Technical Field

[0001] This invention relates to a highly active recombinant type XVII collagen and its rapid preparation method and application, belonging to the field of bioengineering technology. Background Technology

[0002] Collagen is a fibrous, large-molecule protein with excellent biocompatibility and biodegradability, making it one of the most widely used protein materials in biomaterials and regenerative medicine. It is extensively applied in cosmetic fillers, drug delivery systems, surgical sutures, and tissue engineering scaffolds, playing a supporting, repairing, and anti-aging role. Based on differences in protein structure and amino acid composition, human collagen can be divided into 28 types. Type XVII collagen is basement membrane collagen, with a total length of 1497 amino acids, containing three major structural domains: intracellular, transmembrane, and extracellular, comprising 16 non-triple-helix regions and 15 triple-helix regions. As a transmembrane protein on hemidesmosomes, it is primarily responsible for tightly binding the epidermis and dermis, ensuring the integrity of the skin structure.

[0003] Furthermore, type XVII collagen can play an important role in skin wound repair by influencing the migration, proliferation, and differentiation of stem cells. However, type XVII collagen is present in extremely low amounts in humans and animals, and its extraction is extremely difficult. Therefore, biotechnologies such as genetic engineering are typically used to obtain recombinant type XVII collagen.

[0004] Chinese patent document CN117756926A discloses a recombinant type XVII collagen Pro.C17, its preparation method, and its applications. This patent uses *E. coli* as the expression host. Chinese patent document CN118307663A discloses a recombinant type XVII collagen with a triple helix structure and its applications. This patent uses a single plasmid to transform conventional *Pichia pastoris* for production and employs nickel column affinity purification, resulting in high purification costs and making the process difficult to scale up to a large scale.

[0005] Meanwhile, both of these preparation methods result in low yields and high production costs for type XVII collagen. Therefore, there is an urgent need to develop novel recombinant type XVII collagen and a rapid preparation method suitable for industrial production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a highly active recombinant type XVII collagen, its rapid preparation method, and its applications.

[0007] The technical solution of the present invention is as follows:

[0008] A highly active recombinant type XVII collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The nucleotide sequence of the gene encoding the highly active recombinant type XVII collagen is shown in SEQ ID NO.2.

[0010] A recombinant expression vector into which the gene encoding the aforementioned highly active recombinant type XVII collagen is inserted.

[0011] According to a preferred embodiment of the present invention, the expression vector is pPIC9K, pPICZαA, or pGAPCαA.

[0012] A recombinant cell in which the encoding gene or recombinant expression vector of the aforementioned highly active recombinant type XVII collagen is inserted into a host cell.

[0013] According to a preferred embodiment of the present invention, the host cell is Pichia pastoris SMD1163, a protease-deficient yeast.

[0014] The preparation method of the above-mentioned recombinant type XVII collagen includes the following steps:

[0015] (1) The highly active recombinant type XVII collagen encoding gene shown in SEQ ID NO.2 was ligated into expression vectors pPIC9K, pPICZαA and pGAPCαA, respectively, to obtain recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P;

[0016] (2) The recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P obtained in step (1) were sequentially transformed into Pichia pastoris SMD1163 competent cells to obtain recombinant strains.

[0017] (3) After culturing the recombinant strain obtained in step (2), centrifuge and collect the supernatant;

[0018] (4) The supernatant obtained in step (3) is separated and purified to obtain highly active recombinant type XVII collagen.

[0019] According to a preferred embodiment of the present invention, in step (1), the expression vector pGAPCαA is constructed according to the following method:

[0020] Using pNZ8148 plasmid as a template and Cm-F / R as primers, the chloramphenicol resistance gene fragment was amplified by PCR. Then, the expression vector pGAPZαA was double-digested with NcoI and EcoRV restriction enzymes. The amplified chloramphenicol resistance gene fragment was ligated into the double-digested pGAPZαA vector by homologous recombination to obtain the pGAPCαA vector.

[0021] The sequence of primer Cm-F / R is shown below:

[0022] Cm-F:

[0023] 5′-acaaggtgaggaactaaaccATGAACTTTAATAAAATTGATTTAGACAATTGG-3′;

[0024] Cm-R: 5′-tgacataactaattacatgatatcTAAAAACCTTCTTCAACTAACGGG-3′.

[0025] According to a preferred embodiment of the present invention, in step (4), the separation and purification specifically includes:

[0026] The supernatant was first concentrated using a 30 kDa ultrafiltration membrane, and the permeate was collected. Then, it was concentrated using a 1 kDa ultrafiltration membrane, and the retentate was collected to obtain a protein concentrate. Acetone was then added to the protein concentrate under ice bath conditions while stirring until the final acetone concentration was 40%. The resulting mixture was allowed to stand overnight at 4°C, and then centrifuged at 10,000 rpm for 10 min at 4°C to collect the precipitate. The precipitate was reconstituted with ultrapure water and passed through a cation exchange column. The resulting chromatogram was concentrated by ultrafiltration using a 1 kDa membrane, and finally, the concentrate was freeze-dried.

[0027] The above-mentioned highly active recombinant type XVII collagen, and the highly active recombinant type XVII collagen prepared by the above-mentioned method, may be used in any of the following:

[0028] a) Promotes cell adhesion;

[0029] b) Promotes cell proliferation;

[0030] c) Promotes cell migration;

[0031] d) Preparation of cosmetics;

[0032] e) Preparation of medical materials.

[0033] The cell proliferation promoter mentioned above can be used as a basic component of the culture medium to promote cell proliferation, including its application in the culture and preservation of hair follicle stem cells or hair follicle tissue in vitro.

[0034] The cell migration promoter mentioned above can be used as a basic component of the culture medium to promote cell migration, including its application in the culture and preservation of hair follicle stem cells or hair follicle tissue in vitro.

[0035] The cosmetics mentioned include, but are not limited to, skin lotion, skin cream, essence water, face mask, scalp essence, and shampoo.

[0036] The medical materials mentioned include, but are not limited to, filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, and drug delivery carriers, wherein the repair materials include, but are not limited to, bone repair materials, wound dressings, and sutures.

[0037] The beneficial effects of this invention are as follows:

[0038] 1. This invention provides a novel highly active recombinant type XVII collagen protein with good biological activity, possessing excellent cell adhesion, cell proliferation and cell migration promotion effects, and can be compounded with other proteins to become raw materials for tissue engineering products, cosmetics, health products or drugs.

[0039] 2. This invention integrates the highly active recombinant type XVII collagen gene into different locations in the yeast genome using three different vectors, thereby increasing the copy number of the target gene and significantly increasing the expression level of recombinant type XVII collagen, effectively improving the yield of highly active recombinant type XVII collagen.

[0040] 3. This invention optimizes the purification process of recombinant type XVII collagen, improves the purification efficiency of recombinant type XVII collagen, reduces the production cost of recombinant type XVII collagen, increases the production speed of highly active recombinant type XVII collagen, and realizes the rapid preparation of highly active recombinant type XVII collagen. Attached Figure Description

[0041] Figure 1 SDS-PAGE electrophoresis images of Pichia pastoris SMD1163 supernatant (blank), recombinant Pichia pastoris SMD1163-K-COL17A1-P supernatant (single plasmid), and recombinant strain SMD1163-KZG-COL17A1-P supernatant (triple plasmid).

[0042] Figure 2 The plasmid map of the recombinant vector pPIC9K-COL17A1-P.

[0043] Figure 3 The plasmid map of the recombinant vector pPICZαA-COL17A1-P.

[0044] Figure 4 This is a plasmid map of the recombinant vector pGAPCαA-COL17A1-P.

[0045] Figure 5 The images show the lyophilized sponge and electrophoresis results of the highly active recombinant type XVII collagen COL17A1-P of this invention.

[0046] In the figure, A is a lyophilized sponge image of recombinant type XVII collagen COL17A1-P, and B is an electrophoresis image of the lyophilized sponge image of recombinant type XVII collagen COL17A1-P.

[0047] Figure 6 This is a verification result of the cell proliferation-promoting activity of the highly active recombinant type XVII collagen COL17A1-P of this invention.

[0048] Figure 7 This is a verification result of the cell adhesion-promoting activity of the highly active recombinant type XVII collagen COL17A1-P of this invention.

[0049] Figure 8 This is a verification result of the anti-hair loss activity of the highly active recombinant type XVII collagen COL17A1-P of this invention. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. Unless otherwise specified, the reagents and pharmaceuticals involved in the present invention are all commercially available products.

[0051] The protease-deficient Pichia pastoris SMD1163 used in this invention is a common commercially available strain, sold by Invitrogen.

[0052] The expression vectors pNZ8148, pPIC9K, pPICZαA and pGAPZαA used in this invention are conventional plasmid expression vectors, which are available from Invitrogen.

[0053] Example 1: Construction of a novel highly active recombinant type XVII collagen COL17A1-P expression vector

[0054] 1. Design of a novel, highly active recombinant type XVII collagen COL17A1-P

[0055] The full-length sequence of human type XVII collagen was obtained from the NCBI website, accession number KAI4077360.1. Then, the sequence was analyzed using the website (www.ebi.ac.uk / interpro), and an amino acid sequence of a recombinant, highly active type XVII collagen was designed and named COL17A1-P. The specific sequence is as follows:

[0056] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGGPPGPPGPPGPKGDQGQGPPGPPGPQGPKGDKGDPGVPGGQKGEMGTPPGKGDRG PAGPPGHPGP PGPRGHKGEK GDKGDQ (SEQ ID NO. 1).

[0057] 2. Construction of the recombinant vector

[0058] (1) Construction of pPIC9K-COL17A1-P and pPICZαA-COL17A1-P expression vectors

[0059] BGI Genomics Co., Ltd. was commissioned to perform gene fragment analysis on the highly active recombinant type XVII collagen COL17A1-P of this invention according to the nucleotide sequence information shown in SEQ ID NO.2. COL17A1-P The artificial synthesis of ) was then performed using EcoRI and NotI enzymes, respectively. COL17A1-P The expression vectors pPIC9K and pPICZαA were double-digested, and then the digested gene was ligated using T4 DNA ligase. COL17A1-P The recombinant vector pPIC9K-COL17A1-P was constructed by ligating it into two enzyme-digested expression vectors. (Structure shown in Figure 1) Figure 2 As shown), pPICZαA-COL17A1-P (structure as shown) Figure 3 (As shown).

[0060] Among them, double enzyme digestion and ligase ligation are basic molecular biology experimental techniques, which can be referred to in "Molecular Cloning: A Laboratory Manual".

[0061] (2) Construction of pGAPCαA-COL17A1-P expression vector

[0062] Using pNZ8148 plasmid as a template and Cm-F / R as primers, the chloramphenicol resistance gene fragment was amplified by PCR. The sequences of primers Cm-F / R are shown in Table 1. The expression vector pGAPZαA was then double-digested with NcoI and EcoRV restriction enzymes. The amplified chloramphenicol resistance gene fragment was ligated into the double-digested pGAPZαA vector via homologous recombination to obtain the pGAPCαA vector.

[0063] Table 1. Primer sequences

[0064]

[0065] Next, EcoRI and NotI enzymes were used to target the gene, respectively. COL17A1-P The expression vector pGAPCαA was double-digested, and then the digested gene was ligated using T4 DNA ligase. COL17A1-P The recombinant vector pGAPCαA was ligated with the enzyme-digested expression vector pGAPCαA to construct the recombinant vector pGAPCαA-COL17A1-P (structure shown below). Figure 4 (As shown).

[0066] Among them, PCR amplification, double enzyme digestion, homologous recombination ligation and other operations are basic molecular biology experimental techniques, which can be referred to in "Molecular Cloning: A Laboratory Manual".

[0067] Example 2: Construction of recombinant strains

[0068] 1. Constructing the recombinant strain SMD1163-K-COL17A1-P

[0069] The recombinant vector pPIC9K-COL17A1-P described in Example 1 was digested with SalI. After the digested plasmid was recovered, it was electroporated into protease-deficient Pichia pastoris SMD1163 competent cells. The cells were then plated on MD solid medium and cultured upside down at 30°C for 4 days. The resulting single colonies were transferred to YPD solid medium containing 1 mg / mL or 4 mg / mL G418 for screening. The single colonies grown on the 4 mg / mL G418 YPD solid medium were inoculated into 50 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm to obtain recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid).

[0070] MD solid medium formulation: 13.4 g / L yeast basic nitrogen source YNB, 0.4 mg / L biotin, 20 g / L glucose.

[0071] YPD liquid culture medium formula: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose.

[0072] 2. Construction of recombinant strain SMD1163-KZ-COL17A1-P

[0073] Add 1 mL of single-plasmid recombinant Pichia pastoris SMD1163-K-COL17A1-P bacterial culture to 50 mL of fresh YPD liquid medium and incubate at 30 °C and 220 rpm on a shaker until OD500. 6001.3. After standing on ice for 15 min, centrifuge at 4℃ and 1500g for 5 min, discard the supernatant, then add 50 mL of pre-chilled sterile water to resuspend the cells, centrifuge at 4℃ and 1500g for 5 min, discard the supernatant, then add 25 mL of pre-chilled sterile water to resuspend the cells, centrifuge at 4℃ and 1500g for 5 min, discard the supernatant, then add 10 mL of pre-chilled 1 mol / L sorbitol to resuspend the cells, centrifuge at 4℃ and 1500g for 5 min, discard the supernatant, add 1 mL of pre-chilled 1 mol / L sorbitol to resuspend the cells, and aliquot into sterile centrifuge tubes to obtain Pichia pastoris SMD1163-K-COL17A1-P competent cells, and store at -80℃ for later use.

[0074] The recombinant vector pPICZαA-COL17A1-P described in Example 1 was digested with PmeI enzyme. After the digested plasmid was recovered, it was electroporated into Pichia pastoris SMD1163-K-COL17A1-P competent cells. Then, it was plated on YPDZ solid medium containing 100 μg / mL bleomycin and cultured upside down at 30°C for 48 h. The single colonies that grew were transferred to YPDZ solid medium containing 400 μg / mL bleomycin for screening. The single colonies that grew were inoculated into 50 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm to obtain the dual-plasmid recombinant Pichia pastoris SMD1163-KZ-COL17A1-P.

[0075] YPDZ solid culture medium preparation method: Add bleomycin to YPD culture medium at final concentrations of 100 μg / mL and 400 μg / mL respectively.

[0076] 3. Construction of the SMD1163-KZG-COL17A1-P recombinant strain

[0077] Prepare dual-plasmid recombinant Pichia pastoris SMD1163-KZ-COL17A1-P competent cells according to the method obtained in step 2.

[0078] The recombinant vector pGAPCαA-COL17A1-P described in Example 1 was digested with BspHⅠ. After the digested plasmids were recovered, they were electroporated into competent cells of the double-plasmid recombinant Pichia pastoris SMD1163-KZ-COL17A1-P. The cells were then plated on YPDC solid medium containing 25 μg / mL chloramphenicol and cultured upside down at 30°C for 3 days. The resulting single colonies were inoculated into 50 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm to obtain the recombinant strain SMD1163-KZG-COL17A1-P (triple plasmid).

[0079] YPDC culture medium preparation method: Add chloramphenicol to YPD culture medium to a final concentration of 25 μg / mL.

[0080] Example 3: Fermentation of recombinant strains in a fermenter

[0081] 1. The recombinant strain SMD1163-KZG-COL17A1-P constructed in Example 2 was inoculated into YPD liquid medium at a volume ratio of 5%, and cultured in a shaker at 30℃ and 220r / m for 20h to obtain seed liquid.

[0082] 2. Inoculate the seed culture obtained in step 1 into a 10L fully automated fermenter at a 5% (v / v) inoculation rate. Initially, the fermenter contains 4L of BSM Plus liquid culture medium. The temperature is controlled at 30℃, pH at 5.0, stirring speed at 200 rpm, and aeration rate at 10L / min, maintaining dissolved oxygen above 25%. After 18 hours of culture, the initial glycerol is consumed and dissolved oxygen recovers. 50% (w / v) glycerol (containing 12 mL / L PTM1) is added at a rate of 75 g / h, maintaining dissolved oxygen >25% during this period. When the cell wet weight reaches 180 g / L, the glycerol addition is stopped, the fermentation temperature is lowered to 25℃, and the culture is starved for 60 minutes. Then, 100% methanol (containing 12 mL / L PTM1) is added. For PTM1, the methanol flow rate was as follows: first, 12 g / h was added for 2 hours, then 18 g / h for 2 hours, then 24 g / h for 2 hours, and finally 33 g / h until the start of induced fermentation. Timing was started after the methanol was added by gravity, and induced fermentation was carried out for 96 hours (dissolved oxygen was controlled to be >25% during the induced fermentation process). The induced fermentation was then stopped, and the fermentation broth was obtained. After the fermentation was completed, the fermentation broth was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was collected.

[0083] Simultaneously, the protease-deficient Pichia pastoris SMD1163 and the recombinant Pichia pastoris SMD1163-K-COL17A1-P constructed in Example 2 were cultured using the same method, and the supernatant was collected. Then, the supernatant of Pichia pastoris SMD1163 (blank), the supernatant of recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid), and the supernatant of recombinant strain SMD1163-KZG-COL17A1-P (triple plasmid) were subjected to SDS-PAGE electrophoresis. The results are as follows. Figure 1 As shown.

[0084] Depend on Figure 1 It can be seen that the supernatant (blank) of Pichia pastoris SMD1163 does not contain recombinant type XVII collagen COL17A1-P, while the supernatant (single plasmid) of recombinant Pichia pastoris SMD1163-K-COL17A1-P and the supernatant (triple plasmid) of recombinant strain SMD1163-KZG-COL17A1-P contain recombinant type XVII collagen COL17A1-P with a molecular weight of approximately 22 kDa, indicating that the heterologous expression of recombinant type XVII collagen COL17A1-P was successful.

[0085] In this embodiment, the yield of recombinant type XVII collagen COL17A1-P from the supernatant (single plasmid) of recombinant Pichia pastoris SMD1163-K-COL17A1-P was 5.5 g / L, and the yield of recombinant type XVII collagen COL17A1-P from the supernatant (triple plasmid) of recombinant strain SMD1163-KZG-COL17A1-P was 13 g / L. This indicates that the present invention integrates the highly active recombinant type XVII collagen gene into different locations in the yeast genome using three different vectors, increasing the copy number of the target gene, thereby significantly increasing the expression level of recombinant type XVII collagen and effectively improving the yield of recombinant type XVII collagen.

[0086] BSM Plus liquid culture medium formulation: calcium sulfate dihydrate 0.47 g / L, potassium sulfate 9.1 g / L, magnesium sulfate heptahydrate 7.5 g / L, potassium hydroxide 2.1 g / L, glycerol 40 g / L, phosphate 13.4 ml / L, yeast extract 10 g / L, L-histidine 0.5 g / L, antifoaming agent 0.5 g / L.

[0087] Example 4: Rapid purification method for recombinant type XVII collagen COL17A1-P

[0088] The fermentation broth of recombinant Pichia pastoris SMD1163-K-COL17A1-P prepared in Example 3 was collected. The fermentation broth was centrifuged at 10000 rpm for 30 min to obtain the supernatant. The supernatant was first concentrated using a 30 kDa ultrafiltration membrane, and the permeate was collected. Then, it was concentrated again using a 1 kDa ultrafiltration membrane, and the retentate was collected to obtain the protein concentrate. Acetone was then added to the protein concentrate under ice bath conditions with stirring until the final acetone concentration was 40%. The resulting mixture was allowed to stand overnight at 4°C, and then centrifuged at 10000 rpm for 10 min at 4°C to collect the precipitate. The precipitate was reconstituted with ultrapure water and passed through a cation exchange column. The resulting chromatography solution was concentrated using a 1 kDa membrane ultrafiltration membrane. Finally, the concentrate was freeze-dried to obtain purified recombinant type XVII collagen COL17A1-P. The lyophilized sponge and protein electrophoresis images of this recombinant type XVII collagen COL17A1-P are shown below. Figure 5 As shown.

[0089] Depend on Figure 5 As can be seen from the electrophoresis diagram, the molecular weight of the protein is approximately 22 kDa, indicating that this embodiment successfully isolated and purified recombinant type XVII collagen COL17A1-P.

[0090] Example 5: Activity assay of recombinant type XVII collagen COL17A1-P

[0091] 1. Recombinant type XVII collagen COL17A1-P promotes cell proliferation activity.

[0092] Using human immortalized keratinocytes (HaCaT) as the research object, human immortalized keratinocytes (HaCaT) in the exponential growth phase were first collected, counted, and then divided into groups according to a 10-10 ratio. 4 Cells were seeded into 96-well plates with 5 replicates. After culturing for 24 hours, 1 mg / mL of recombinant type XVII collagen COL17A1-P prepared in Example 4 was added to each well. After culturing for another 48 hours, the original culture medium was aspirated, and the cells were washed once with PBS. Fresh culture medium was added, and 10 mL of CCK8 (a highly sensitive, non-radioactive colorimetric assay used to determine the number of viable cells in cell proliferation or toxicity experiments) was added to each well. After incubation at 37°C for 1 hour, the absorbance of each well was measured using a multi-mode microplate reader at a wavelength of 450 nm. Cell viability in each group was statistically analyzed. Commercially available bovine serum albumin (BSA) and commercially available recombinant type XVII collagen were used as controls. The experimental results are as follows: Figure 6 As shown.

[0093] Depend on Figure 6 It can be seen that the recombinant type XVII collagen COL17A1-P of the present invention showed a trend of promoting HaCaT cell activity within the concentration gradient of 0.25~4 mg / mL, with no toxic effect, and the differences were statistically significant. This indicates that the recombinant type XVII collagen COL17A1-P of the present invention has better HaCaT cell proliferation-promoting activity than the control group BSA and commercially available recombinant type XVII collagen, and is a highly active recombinant type XVII collagen COL17A1-P.

[0094] 2. Cell adhesion activity of recombinant type XVII collagen COL17A1-P

[0095] The purified lyophilized recombinant type XVII collagen COL17A1-P prepared in Example 4, commercially available bovine serum albumin (BSA) as a control, and commercially available recombinant type XVII collagen were dissolved in pure water. Protein concentration was determined using the empirical formula for UV protein quantification: C (mg / mL) = 0.144 × (A215 - A225). The solution was then diluted to 0.5 mg / mL with PBS buffer (pH=7.4). 100 μL of each protein solution to be tested was added to a 96-well cell culture plate, with blank PBS buffer as a control. The plate was incubated at room temperature for 60 min. Then, 10 μL of each protein solution was added to each well. 5 NIH / 3T3 cells in good culture condition were incubated at 37°C and 5% CO2 for 60 min. Cells in the wells were washed four times with PBS buffer. Cell migration activity was assessed by measuring absorbance at OD=492 nm using an LDH detection kit. Results are shown below. Figure 7As shown, the absorbance at OD=492nm corresponds to the cell adhesion activity of the collagen sample: the higher the absorbance, the more cells the protein adheres to, and the higher the adhesion activity.

[0096] Depend on Figure 7 It is known that the cell adhesion activity of the recombinant XVII type collagen COL17A1-P of the present invention is superior to that of BSA and commercially available recombinant XVII type collagen, indicating that it can help cells adhere to the cell wall or adhere to the extracellular matrix in a short time, which is more conducive to building a better extracellular environment. It is a highly active recombinant XVII type collagen COL17A1-P.

[0097] 3. Recombinant XVII type collagen COL17A1-P's anti-hair loss effect

[0098] The recombinant type XVII collagen COL17A1-P concentrate prepared in Example 4 (Group A), the commercially available recombinant type XVII collagen essence matrix control (Group B), and minoxidil tincture (Group C, minoxidil tincture is a peripheral vasodilator approved by the National Medical Products Administration, which can stimulate hair growth in patients with male pattern baldness and alopecia areata when used topically) were used as test samples to test their anti-hair loss effects on humans.

[0099] Test groups: Group A, 11 participants, 11 valid samples completed. 6 were female and 5 were male, aged 34-50 years, with a mean age of 41.7±4.9 years. Group B, 11 participants, 11 valid samples completed. 6 were female and 5 were male, aged 24-54 years, with a mean age of 41.8±8.1 years. Group C, 11 participants, 11 valid samples completed. 7 were female and 4 were male, aged 26-49 years, with a mean age of 39.9±6.8 years. All participants had hair lengths between 5 and 40 cm, experienced significant hair loss and mild thinning, and had more than 10 hairs lost after 60 combing tests and still more than 10 hairs after a 2-week shampooing period, meeting the project requirements.

[0100] Test Procedure: For the recombinant type XVII collagen essence and its matrix control: use one bottle daily, 2-3 times daily. Tear open the bottle opening in the direction indicated by the aluminum seal, attach the dropper, part the hair, and apply evenly to the scalp, gently massaging to promote absorption. No rinsing is required after use. For the minoxidil solution: apply 1 mL (approximately 7 sprays) once daily to the affected area of ​​the scalp, starting from the center and massaging for 3-5 minutes. Use this dosage regardless of the size of the affected area, with a daily total not exceeding 2 mL. Test results are as follows: Figure 8 As shown.

[0101] Depend on Figure 8 It can be seen that with the extension of the usage time, the amount of hair loss in Experiment A group decreased significantly, indicating that the recombinant XVII type collagen COL17A1-P of the present invention has the effect of slowing down the reduction of hair and preventing hair loss.

Claims

1. A highly active recombinant collagen type XVII, characterized in that, The amino acid sequence is shown as SEQ ID NO.

1.

2. The gene encoding the highly active recombinant collagen type XVII according to claim 1, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The gene coding the high-activity recombinant collagen XVII of claim 2 is inserted into an expression vector.

4. The recombinant expression vector of claim 3, wherein, The expression vector is pPIC9K, pPICZαA or pGAPCαA.

5. A recombinant cell, characterized in that, The gene coding the high-activity recombinant collagen XVII of claim 2 or the recombinant expression vector of claim 3 is inserted into a host cell.

6. The recombinant cell of claim 5, wherein The host cell is protease-deficient Pichia pastoris SMD1163.

7. The method for preparing the highly active recombinant type XVII collagen according to claim 1, characterized in that, The method comprises the following steps: (1) connecting the gene coding the high-activity recombinant collagen XVII shown as SEQ ID NO. 2 to expression vectors pPIC9K, pPICZαA and pGAPCαA respectively to obtain recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P; (2) transforming the recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P obtained in step (1) into Pichia pastoris SMD1163 competent cells in turn to obtain recombinant strains; (3) culturing the recombinant strains obtained in step (2), centrifuging and collecting the supernatant; (4) separating and purifying the supernatant obtained in step (3) to obtain the high-activity recombinant collagen XVII.

8. The production method according to claim 7, wherein In step (4), the expression vector pGAPCαA is constructed as follows: A chloramphenicol resistance gene fragment is obtained by PCR amplification using pNZ8148 plasmid as a template, then the expression vector pGAPZαA is double digested with Nco I and Eco RV endonuclease, the amplified chloramphenicol resistance gene fragment is connected to the double-digested pGAPZαA vector by homologous recombination to obtain the pGAPCαA vector.

9. The production method according to claim 7, wherein In step (4), the separation and purification are specifically as follows: The supernatant is first concentrated by a 30 kDa ultrafiltration membrane, and the permeate is collected; then the supernatant is concentrated by a 1 kDa ultrafiltration membrane, and the retentate is collected to obtain a protein concentrate; then acetone is added to the protein concentrate under ice bath conditions, and the mixture is stirred to obtain a final acetone content of 40%, the obtained mixture is placed at 4°C overnight, and then centrifuged at 10000 rpm for 10 min at 4°C to collect the precipitate; the precipitate is redissolved with ultrapure water and passed through a cation exchange column, the eluate is concentrated by a 1 kDa membrane ultrafiltration, and finally the concentrate is freeze-dried.

10. The high-activity recombinant collagen XVII obtained by the preparation method of claim 7 is used in any one of the following applications: a) preparing cosmetics; b) preparing medical materials.

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