Recombinant vector for expressing natural long-sequence recombinant collagen type XVII and preparation method and application thereof

Abstract

The present application relates to a kind of expression natural long sequence recombinant collagen type XVII and preparation method and application, it is related to protein field, it includes the nucleotide sequence of the nucleotide sequence of the recombinant collagen type XVII of coding, optionally the nucleotide sequence of the recombinant collagen type XVII of coding has at least one nucleotide sequence of the amino acid sequence of coding as shown in SEQ ID NO:1 (optionally at least one nucleotide sequence as shown in any one of SEQ ID NO:2-4).The target protein produced by the present application is excellent in safety performance, and shows great potential in multiple application scenarios such as plastic beauty, hair regeneration, etc., and is expected to break through the past difficulties, truly open a new chapter of commercial application of recombinant COL17 protein, and inject new vitality into related industries.

Description

Technical Field

[0001] This invention relates to the field of proteins, and more particularly to recombinant vectors for expressing natural long-sequence recombinant type XVII collagen, their preparation methods, and applications. Background Technology

[0002] In the current booming development of modern medicine and the beauty industry, skin-related research is becoming increasingly in-depth, with type XVII collagen (COL17) protein becoming a focus of attention. As an indispensable key component of the skin's basement membrane, COL17 plays a crucial role in maintaining the normal physiological structure and function of the skin. The type XVII collagen α1 chain (COL17A1) is composed of three α1 chains with identical amino acid sequences. The mature peptide of each α1 chain consists of 1497 amino acids. Its unique transmembrane structure gives it the ability to anchor cells, while the collagen triple helix structure provides a stable supporting framework for the skin's basement membrane.

[0003] However, the research and application of COL17 protein in reality faces many thorny challenges. On the one hand, the amount of COL17 protein in animals is extremely scarce, which drastically increases the difficulty of obtaining this natural raw material. Faced with such a minute quantity and complex distribution of protein, traditional extraction methods are inadequate and simply cannot meet the needs of large-scale production. On the other hand, the field of recombinant proteins also encounters difficulties. Logically, the more complete the protein sequence, the more ideal its function should be; however, this is not the case in reality. As the length of recombinant protein sequences increases, stability decreases rapidly, much like the increasing instability of a tall building as the floors rise. The consequence of this instability is difficulty in efficient purification using conventional techniques, resulting in very low yields and ultimately causing production costs to skyrocket to unacceptable levels, severely hindering commercialization. Moreover, due to their large molecular weight, long-chain proteins are prone to higher-order structural changes such as folding and entanglement during recombinant expression. These changes may have a counterproductive effect on their biological activity, hindering their commercial application in various fields.

[0004] Taking all factors into consideration, the key technological bottleneck in this field has become apparent: how to overcome numerous obstacles to achieve efficient and stable expression of the 585-amino acid natural long sequence of COL17A1 in host cells while preserving its biological activity. Clearly, if this problem is not properly resolved, many potential medical and cosmetic applications of COL17 will remain theoretical and unable to truly benefit patients and consumers. For example, severe skin diseases such as epidermolysis bullae are caused by the absence or mutation of the COL17 protein. Achieving stable mass production of this protein could potentially open up new avenues for the treatment of these diseases. In the cosmetic field, its potential effects on skin repair and hair regeneration are also attracting the attention of numerous researchers and practitioners.

[0005] Selecting the most suitable production sequence that balances functionality, expression level, and stability is a research challenge in this field. Summary of the Invention

[0006] Technical issues

[0007] In view of this, the technical problem to be solved by this invention is how to provide a recombinant vector for expressing natural long-sequence recombinant type XVII collagen, its preparation method, and its applications. This invention, through massive screening, successfully screened a long fragment of 585 amino acids from the natural COL17A1 sequence. Based on this, an engineered bacterium capable of efficiently and stably expressing this specific sequence was further constructed. Through this series of innovative measures, verification experiments showed that the produced target protein exhibits excellent safety performance and demonstrates great potential in multiple application scenarios such as cosmetic surgery and hair regeneration. It is expected to break through past limitations and truly open a new chapter in the commercial application of recombinant COL17 protein, injecting new vitality into related industries.

[0008] Solution

[0009] To solve the above technical problems, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a recombinant vector for expressing natural long-sequence recombinant type XVII collagen, comprising a nucleotide sequence encoding recombinant type XVII collagen, optionally having at least one segment encoding an amino acid sequence as shown in SEQ ID NO:1.

[0011] The 585 amino acid sequence of the natural long COL17A1 sequence of the recombinant type XVII collagen is shown in SEQ ID NO:1:

[0012] .

[0013] Furthermore, the nucleotide sequence encoding recombinant type XVII collagen has at least one nucleotide sequence as shown in any one of SEQ ID NO:2 to 4, and optionally has at least one nucleotide sequence of the amino acid sequence shown in the nucleotide sequence shown in SEQ ID NO:2 or 3.

[0014] The nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:1 are shown in SEQ ID NO:2, 3, or 4, wherein the optimized codon is shown in SEQ ID NO:2:

[0015]

[0016] A nucleotide sequence containing an enzyme cleavage site, a start codon, and a stop codon can be shown as SEQ ID NO:3:

[0017]

[0018] The natural codon is shown in SEQ ID NO:4:

[0019]

[0020] Optionally, the backbone vector is the pGAPZ A-αA expression vector.

[0021] In a second aspect, a microorganism comprising the recombinant vector described in the first aspect is provided.

[0022] Further, the microorganism is Pichia pastoris, and optionally, the Pichia pastoris is Pichia pastoris GS115.

[0023] Thirdly, a method for preparing naturally expressed long-sequence recombinant type XVII collagen is provided, comprising the following steps:

[0024] The recombinant vector described in the first aspect is transferred into yeast to obtain genetically engineered yeast, or the microorganism described in the second aspect is added to a culture medium for fermentation, the fermentation broth is collected, and the COLXVII type collagen is purified.

[0025] Furthermore, the culture medium used was YPD medium and trace elements;

[0026] Optionally, the trace elements in the culture medium include the following components in parts by weight: boric acid 0.01–0.025 g / L, copper sulfate pentahydrate 4–5 g / L, manganese sulfate 1–2 g / L, ferrous sulfate 45–50 g / L, riboflavin 0.25–0.4 g / L, pyridoxal phosphate 0.7–1.1 g / L, nicotinic acid 0.4–0.5 g / L, thiamine 0.22–0.25 g / L, and biotin 0.008–0.014 g / L;

[0027] Optionally, the trace elements in the culture medium include the following components in parts by weight: boric acid 0.025 g / L, copper sulfate pentahydrate 4 g / L, manganese sulfate 2.0 g / L, ferrous sulfate 45 g / L, riboflavin 0.4 g / L, pyridoxal phosphate 0.7 g / L, nicotinic acid 0.5 g / L, thiamine 0.22 g / L, and biotin 0.014 g / L.

[0028] The culture medium can be supplemented with the following components as needed: glycerol, methanol, glucose, yeast extract, plant peptone, ammonium sulfate, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, zinc sulfate, copper sulfate, proline, lysine, glycine, citric acid, disodium hydrogen phosphate, sorbitol, and Tween-80. Example combinations include: methanol, glucose, yeast extract, plant peptone, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, zinc sulfate, copper sulfate, proline, lysine, citric acid, disodium hydrogen phosphate, and sorbitol.

[0029] Optionally, the purification method includes: taking the fermentation culture broth and removing cell debris by centrifugation and ultrafiltration, clarifying the culture broth with hollow fiber, purifying it by ion exchange chromatography, and further separating, concentrating and purifying to obtain the target protein.

[0030] Fourthly, a long-sequence recombinant type XVII collagen obtained using the preparation method described in the third aspect is provided.

[0031] Fifthly, the application of natural long-sequence recombinant type XVII collagen prepared by the recombinant vector described in the first aspect, the microorganism described in the second aspect, or the preparation method described in the third aspect in the preparation of skin repair dressings, medical aesthetic injection products, hair regeneration products, implants, biomaterials, and medical devices.

[0032] Furthermore, the hair regeneration product is a hair regeneration product that promotes the proliferation of keratinocytes or other biomedical materials used in the development of medical device products.

[0033] Beneficial effects

[0034] (1) This invention has successfully constructed an efficient and stable expression system through amino acid sequence selection / codon optimization and fine regulation of the expression system. This system can express the target protein in large quantities in Pichia pastoris host, providing technical support for the large-scale production of natural long sequences of type XVII collagen, and has broad application prospects in the fields of plastic surgery and hair regeneration.

[0035] (3) Through massive screening, this invention successfully identified a long fragment of 585 amino acids from the natural COL17A1 sequence. Based on this, an engineered bacterium capable of efficiently and stably expressing this specific sequence was further constructed. Through this series of innovative measures, verification experiments showed that the produced target protein exhibited excellent safety performance and demonstrated great potential in multiple application scenarios such as cosmetic surgery and hair regeneration. It is expected to break through past difficulties and truly open a new chapter in the commercial application of recombinant COL17 protein, injecting new vitality into related industries.

[0036] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, and to make the above and other objects, technical features and advantages of the present invention easier to understand, one or more preferred embodiments are listed below and described in detail with reference to the accompanying drawings. Attached Figure Description

[0037] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0038] Figure 1 A schematic diagram of the recombinant COL17A1 plasmid in Example 1 of the present invention;

[0039] Figure 2 Electrophoresis diagram of the enzyme digestion results of recombinant COL17A1 plasmid-2 in Example 1 of the present invention.

[0040] Figure 3 DNA sequencing results of recombinant COL17A1 plasmid-2 in Example 1 of the present invention.

[0041] Figure 4 SDS gel electrophoresis image of the fermentation broth of recombinant COL17A1 plasmid-2 in Example 2 of the present invention, wherein the plasmid represents recombinant COL17A1 plasmid-2, and single enzyme digestion represents single enzyme digestion of the plasmid.

[0042] Figure 5 The results of the difference in the proliferative effect of recombinant COL17A1 collagen on keratinocytes before and after codon optimization in Test Example 1 of this invention. (Experimental Group 1: Recombinant collagen fermented without codon optimization; Experimental Group 2: Recombinant collagen fermented after codon optimization; *P<0.05, **P<0.01, n=6)

[0043] Figure 6 The effect of the 585-amino acid recombinant COL17A1 natural long-sequence collagen in Test Example 3 of this invention on promoting hair regeneration in mice (21 days). Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, methods, means, etc., well-known to those skilled in the art, are not described in detail in order to highlight the spirit of the present invention.

[0046] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0047] This invention verifies the safety of the recombinant 585-amino acid natural long sequence COL17A1 through in vitro cell and animal experiments. Its biological activity is also verified through in vitro cell experiments and animal model experiments. For example, cell adhesion, cell proliferation, and cell migration experiments are conducted to assess the effects of the 585-amino acid COL17A1 natural long sequence on cell growth and function; dermal injection experiments in model animals are conducted to assess the potential hair regeneration-promoting effect of the 585-amino acid COL17A1 natural long sequence.

[0048] Example 1. Gene Synthesis and Vector Construction

[0049] 1) Construction of optimized codon gene elements: The coding sequence is tandemly arranged as follows: Kpn I + ATG + nucleotide sequence encoding COL17A1 585AA (SEQ ID NO:1) + TAA + Not I, wherein the KpnI restriction site is GGTACC, the Not I restriction site is GCGGCCGC, ATG corresponds to the start codon, and TAA corresponds to the stop codon. The nucleotide sequence encoding COL17A1 585AA (SEQ ID NO:1) uses the optimized codon as shown in SEQ ID NO:2. The constructed gene element is shown in SEQ ID NO:3. Nucleotide elements containing the natural codon as shown in SEQ ID NO:4 were also synthesized simultaneously (the difference from the optimized codon gene element is that the restriction sites are replaced with NotI and SalI (GTCGAC) respectively). Nucleotide elements are artificially synthesized, for example, by Shenzhen BGI Genomics Co., Ltd.

[0050] 2) The above gene elements and pGAPZ A-αA vector were digested with KpnI and NotI enzymes, respectively, and then ligated using T4 DNA ligase to obtain the recombinant COL17A1 plasmid (e.g., Figure 1 As shown in the image, the plasmids were named recombinant COL17A1 plasmid-1 (natural codons before optimization) and recombinant COL17A1 plasmid-2 (natural codons after optimization) based on whether the codons were optimized. These plasmids were transformed into *E. coli* DH5α competent cells, and positive clones were screened. The integration of the gene into the positive clones was confirmed by PCR and enzyme digestion analysis. Plasmid DNA was extracted, and DNA sequencing was used to further verify the successful insertion of the target gene.

[0051] The results of Not I digestion of plasmid-2, COL17A1, are as follows: Figure 2 DNA sequencing results are as follows Figure 3 The results showed that recombinant COL17A1 plasmid-1 and recombinant COL17A1 plasmid-2 were successfully constructed.

[0052] The pGAPZ A-αA vector selected in this invention contains the GAP promoter and is suitable for Pichia pastoris expression systems, allowing for expression without inducers.

[0053] Example 2: Protein Expression, Purification and Detection

[0054] Recombinant COL17A1 plasmid-1 and recombinant COL17A1 plasmid-2 were transformed into Pichia pastoris GS115 host cells via electroporation. The transformed cells were then screened for antibiotics (using G418 resistance in YPD medium) to obtain positive clones. The fermentation supernatant was analyzed by SDS-PAGE gel electrophoresis. Western blot analysis was performed using a specific antibody against COL17 to determine the expression level of the target protein.

[0055] SDS gel electrophoresis detection and analysis, such as Figure 4 The results showed that the recombinant COL17A1 plasmid-2 was successfully constructed and could express the target protein.

[0056] Protein expression:

[0057] The fermentation process for producing recombinant collagen in a 5L fermenter (brand and model: XAN-Ferment-5L): Two strains of Pichia pastoris (before and after codon optimization) were picked from glycerol tubes and inoculated onto slant agar plates containing suitable culture medium. They were incubated at 28℃ for 48 hours until single colonies appeared. Then, single colonies were picked and inoculated into 300mL shake flasks containing liquid culture medium, placed on a shaker, and incubated at 28℃ and 250rpm for 72 hours to bring the cells into the logarithmic growth phase and achieve a cell density OD0.05. 600 At 3 PM, inoculation was carried out by transferring 8% of the seed culture from the seed tank into a 5L fermenter. A suitable basal medium (YPD medium + trace element formula 3) was used, with the temperature controlled at 28℃ and pH 5.0-6.0. Dissolved oxygen was maintained at 30%-40% by adjusting the stirring speed and aeration rate. Fed culture phase: When the carbon source and other nutrients in the basal medium were depleted to a certain extent, fed culture medium was started. The feeding rate was adjusted according to cell growth and metabolism to maintain cell growth and product synthesis. Simultaneously, parameters such as dissolved oxygen and pH were closely monitored and adjusted as needed. Cell density (OD) was monitored throughout the culture process. 600After approximately 68 hours, the cell density stopped increasing. After being placed in a container, the fermentation broth was processed and the expression level of the target protein was detected.

[0058] The expression levels of the target protein before and after codon optimization are shown in Table 1:

[0059] Table 1. Comparison of protein expression levels before and after optimization of the natural long codon sequence of 585 recombinant COL17A1.

[0060]

[0061] As shown in Table 1, the optimized codons used in this invention can effectively increase the expression level of the target protein by 2.84 times.

[0062] In this embodiment, the composition of the culture medium and the formulation of trace elements were also optimized, and the results are shown in Table 2.

[0063] Table 2. Effects of trace elements on the expression level of recombinant COL17A1 natural long sequence (5L fermenter)

[0064]

[0065] The trace element composition of each formula in Table 2 is as follows:

[0066] Formula 1: Boric acid 0.015g / L, copper sulfate pentahydrate 3g / L, manganese sulfate 1.5g / L, ferrous sulfate 35g / L, riboflavin 0.35g / L, pyridoxal phosphate 0.85g / L, nicotinic acid 0.6g / L, thiamine 0.2g / L and biotin 0.01g / L.

[0067] Formula 2: Boric acid 0.02g / L, copper sulfate pentahydrate 2.5g / L, manganese sulfate 1.2g / L, ferrous sulfate 40g / L, riboflavin 0.3g / L, pyridoxal phosphate 1.0g / L, nicotinic acid 0.7g / L, thiamine 0.18g / L, biotin 0.012g / L.

[0068] Formula 3: Boric acid 0.025g / L, copper sulfate pentahydrate 4g / L, manganese sulfate 2.0g / L, ferrous sulfate 45g / L, riboflavin 0.4g / L, pyridoxal phosphate 0.7g / L, nicotinic acid 0.5g / L, thiamine 0.22g / L, biotin 0.014g / L.

[0069] Formula 4: Boric acid 0.01g / L, copper sulfate pentahydrate 5g / L, manganese sulfate 1g / L, ferrous sulfate 50g / L, riboflavin 0.25g / L, pyridoxal phosphate 1.1g / L, nicotinic acid 0.4g / L, thiamine 0.25g / L, biotin 0.008g / L.

[0070] Formula 5: Boric acid 0.03g / L, copper sulfate pentahydrate 2g / L, manganese sulfate 2.2g / L, ferrous sulfate 30g / L, riboflavin 0.45g / L, pyridoxal phosphate 0.6g / L, nicotinic acid 0.8g / L, thiamine 0.15g / L, biotin 0.015g / L.

[0071] Protein purification process:

[0072] Purification of 585 amino acid-rich recombinant COL17A1 natural long-sequence collagen from fermentation broth:

[0073] 1) Cell harvesting: Cells in the culture medium are collected by centrifugation to remove cellular impurities.

[0074] 2) Hollow fiber clarification: The hollow fiber filtration system is used to remove larger cell debris and undissolved impurities, thus clarifying the culture medium.

[0075] 3) Ultrafiltration concentration: Proteins are further concentrated through an ultrafiltration membrane to remove small molecule impurities.

[0076] 4) Ion exchange chromatography: The recombinant protein was purified using an ion exchange chromatography column, and the target protein was collected and confirmed by mass spectrometry analysis.

[0077] 5) Separation and concentration: Depending on the needs, size exclusion chromatography (GPC) can be used to further purify the protein, remove any low molecular weight impurities, and finally obtain recombinant protein with a purity of over 99%.

[0078] Validation method: BCA method and SDS-PAGE were used to analyze and confirm the content, purity and molecular weight of the target protein.

[0079] Test Example 1: Verify the difference in the proliferative effect of 585 amino acid recombinant COL17A1 natural long-sequence collagen on keratinocytes before and after codon optimization.

[0080] 1) Cell resuscitation and culture: The keratinocyte cell line was taken out from liquid nitrogen and quickly placed in a 37°C water bath for resuscitation. Then it was transferred to a culture flask containing culture medium (DMEM medium containing 10% fetal bovine serum) and placed in a cell culture incubator for culture. When the cell confluence reached 80%-90%, it was passaged.

[0081] (2) Cell seeding: Keratinocytes in the logarithmic growth phase were digested with trypsin to prepare a cell suspension, and the cells were counted and the cell density was adjusted to 5 × 10⁶ cells / year. 3 -1×10 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated for 24 h to allow the cells to adhere to the plate.

[0082] (3) Treatment of test substance: Cells were divided into 7 groups: a blank control group and 6 experimental groups, with 6 wells in each group. Three experimental groups contained low, medium, and high doses of uncodon-optimized recombinant collagen fermented, while the other three groups contained low, medium, and high doses of codon-optimized recombinant collagen fermented. The control group used culture medium without the test substance, while the experimental groups were treated with a certain amount of sterile recombinant collagen test substance, resulting in concentrations of 1 mg / mL, 2 mg / mL, and 5 mg / mL in the culture medium of the two experimental groups, respectively. Each group had 6 replicates.

[0083] (4) Culture and detection: Place the 96-well plate in an incubator and continue culturing. Detect the cells after 24 hours of culture (excessive culture time can lead to cell confluence and inaccurate results). Before detection, add 10 μL of CCK-8 reagent to each well and continue incubation for 2 hours. Then, use a microplate reader to measure the absorbance (OD value) at 450 nm.

[0084] (5) Data processing: Calculate the cell proliferation rate of each group:

[0085] Proliferation rate (%) = (OD value of experimental group - OD value of control group) / OD value of control group × 100%.

[0086] Statistical software was used to compare the effects of different test substances at the same concentration on the proliferation rate of keratinocytes at the same time point using one-way ANOVA data.

[0087] Experimental results are as follows Figure 5 As shown, after three cellular experiments, the results showed that the 585-amino acid recombinant COL17A1 natural long sequence after codon optimization had a significantly greater effect on the proliferation of keratinocytes than before optimization (P < 0.05). This may be because codon optimization affects protein folding and thus biological function. This functional enhancement is of great significance for the commercialization of related products in the future.

[0088] Test Example 2: Evaluation of the oral safety of the 585-amino acid recombinant COL17A1 natural long sequence (oral acute toxicity test).

[0089] Twenty-four adult healthy Kunming mice (6-8 weeks old, weighing 20-25g, half male and half female) were purchased from Beijing Jinmuyang Experimental Animal Breeding Co., Ltd. They were randomly divided into four groups of six mice each: a blank control group and three experimental groups receiving low, medium, and high doses of recombinant COL17A1 natural long-sequence collagen (585 amino acids), at doses of 1000 mg / kg, 5000 mg / kg, and 15000 mg / kg, respectively.

[0090] Before conducting acute toxicity tests, experimental animals are housed in the animal facility for 1-2 days to allow them to fully acclimatize to the laboratory environment. After observation confirms their health, they are randomly assigned to groups. Before the test, experimental animals are fasted overnight, generally for about 16 hours, but water intake is not restricted. The test substance is administered orally by gavage, and observations are conducted at 7 and 14 days after oral administration. If animals continue to die after 4 days, the observation period needs to be extended to 14 days, and if necessary, to 28 days. The number of deaths, time of death, and symptoms of poisoning are recorded. If any animals die, the LD50 of the test substance is determined using the Horn method. 50 value.

[0091] Oral LD50 in mice 50 The corresponding lethal dose classifications for humans are shown in Table 3. The experimental results are shown in Table 4.

[0092] Table 3 Acute toxicity (LD50) dose classification

[0093]

[0094] Table 4. Results of acute oral toxicity test of 585 amino acid recombinant COL17A1 natural long sequence in mice.

[0095]

[0096] The results in Table 4 show that the 585-amino acid recombinant COL17A1 natural long-sequence recombinant COL17A1 collagen prepared in this invention has high safety and is non-toxic.

[0097] Test Example 3: Functional Validation (Hair Regeneration Promotion Experiment in Mice)

[0098] Experimental protocol: Four to five-week-old C57BL / 6 mice (purchased from Beijing Jinmuyang Experimental Animal Breeding Co., Ltd.) were selected and randomly divided into groups after one week of acclimatization. The hair on the backs of the mice was gently shaved with an electric clipper, followed by depilatory cream to induce the hair follicles into the resting phase. The model group was then subcutaneously injected with testosterone propionate or dihydrotestosterone (1-2 mg / kg body weight) into the neck and back, while the control group received an equal volume of the solvent subcutaneously. This was done once daily for approximately four weeks. The skin at the shaved area on the backs of the mice was photographed weekly, and the hair growth was recorded and scored. After successful model establishment, 50 mice were selected and divided into 5 groups, with 10 mice randomly assigned to each group. The mice were divided into a model group, a positive control group (minoxidil solution), and low-, medium-, and high-dose experimental groups of 585-amino acid recombinant COL17A1 natural long-sequence collagen, with doses of 1 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. In the model group, an equal volume of physiological saline was applied to the hairless area on the back of the mice. In the positive control group, an appropriate amount of minoxidil solution was applied. In the experimental groups, the skin of the mice was treated with a 0.5 mm roller, followed by application of the 585-amino acid recombinant COL17A1 natural long-sequence collagen solution, once daily. Hair growth was observed and recorded at 7, 14, and 21 days after administration of the test substance. Photos were taken, and the number of mice with hair regrowth exceeding half of the hairless skin area was calculated and recorded.

[0099] The results are as follows Figure 6 As shown in Table 5, the results indicate that the 585-amino acid recombinant COL17A1 natural long-sequence collagen of the present invention promotes hair regeneration, and the hair grows evenly with color and condition consistent with healthy hair. The growth-promoting effect increases with increasing concentration.

[0100] Table 5. Results of experiments on hair regeneration promotion using the natural long sequence of recombinant COL17A1 containing 585 amino acids.

[0101]

[0102] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. Any simple modifications, equivalent changes, and alterations made to the foregoing exemplary embodiments should fall within the scope of protection of the present invention.

Claims

1. The application of a recombinant vector expressing natural long-sequence recombinant type XVII collagen in the preparation of hair regeneration products, wherein the recombinant vector comprises a nucleotide sequence encoding recombinant type XVII collagen, and the nucleotide sequence encoding recombinant type XVII collagen is as shown in SEQ ID NO:2 or 3; The hair regeneration product is a hair regeneration product that promotes the proliferation of keratinocytes.

2. The application according to claim 1, characterized in that, The backbone vector for the recombinant vector is the pGAPZ A-αA expression vector.

3. The application according to claim 1, characterized in that, The recombinant vector expressed the target protein in the Pichia pastoris host.

4. The application according to claim 3, characterized in that, The Pichia pastoris is Pichia pastoris GS115.

5. The application according to claim 3, characterized in that, A method for preparing naturally expressed long-sequence recombinant type XVII collagen includes the following steps: The recombinant vector was transferred into yeast to obtain genetically engineered yeast, which was then added to a culture medium for fermentation. The fermentation broth was collected and purified to obtain recombinant type XVII collagen.

6. The application according to claim 5, characterized in that, The culture medium used was YPD medium and trace elements.

7. The application according to claim 6, characterized in that, The trace elements in the culture medium include the following components by weight: boric acid 0.01~0.025 g / L, copper sulfate pentahydrate 4~5 g / L, manganese sulfate 1~2 g / L, ferrous sulfate 45~50 g / L, riboflavin 0.25~0.4 g / L, pyridoxal phosphate 0.7~1.1 g / L, nicotinic acid 0.4~0.5 g / L, thiamine 0.22~0.25 g / L, and biotin 0.008~0.014 g / L.

8. The application according to claim 6, characterized in that, The trace elements in the culture medium include the following components by weight: boric acid 0.025 g / L, copper sulfate pentahydrate 4 g / L, manganese sulfate 2.0 g / L, ferrous sulfate 45 g / L, riboflavin 0.4 g / L, pyridoxal phosphate 0.7 g / L, nicotinic acid 0.5 g / L, thiamine 0.22 g / L, and biotin 0.014 g / L.

9. The application according to any one of claims 5 to 8, characterized in that, The purification method includes: taking the fermentation culture medium and removing cell debris by centrifugation and ultrafiltration, clarifying the culture medium with hollow fiber, purifying it by ion exchange chromatography, and further separating, concentrating and purifying to obtain the target protein.

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