A recombinant humanized collagen type xvii and uses thereof

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to a recombinant humanized collagen type XVII and application thereof. The recombinant humanized collagen type XVII provided by the present application is obtained by selecting two fragments of human collagen type XVII with GPP at both ends, connecting them in turn, and performing 8 times of tandem repeats. The recombinant humanized collagen type XVII prepared by the present application not only has the advantages of strong stability, high expression and good hydrophilicity, but also can significantly up-regulate the expression of VEGF gene and WNT10B gene, and has the effect of preventing hair loss. In addition, the amino acid sequence of the recombinant humanized collagen type XVII prepared by the present application is 100% identical to the corresponding part of natural human collagen type XVII, and the application thereof in human body will not cause immune rejection, and has high safety.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant type XVII humanized collagen and its applications. Background Technology

[0002] Collagen is the most abundant protein in the human body, containing multiple types and accounting for 25-30% of total protein. Collagen is distributed in various parts of the body, including skin, tendons, cornea, cartilage, and blood vessels, providing structural support and maintaining the normal shape of various organs and tissues. Type XVII collagen is a transmembrane protein containing 1497 amino acids, composed of an extracellular, transmembrane, and intracellular region. The extracellular region contains 15 collagen domains and 16 non-collagen domains. Besides its role in repairing the basement membrane and promoting epidermal turnover, type XVII collagen is also a component of hemidesmosomes in cells and plays an important role in hair growth.

[0003] In recent years, many people are facing hair loss problems due to high life pressure, poor work and rest, scalp inflammation, aging, malnutrition and other reasons. Studies have shown that the consumption of type XVII collagen in hair follicle stem cells is a key factor leading to hair follicle atrophy and hair loss (Matsumura H, Mohri Y, Binh TN, et al. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis[J]. Science, 2016, 351(6273):aad4395).

[0004] Currently, the main sources of collagen include extraction from animal tissues and preparation using recombinant DNA technology. While collagen can be extracted from animal tissues through acid and alkali hydrolysis, this type of collagen is a heterologous protein and may pose risks such as immune rejection, allergies, and disease. The development of recombinant technology allows for the high-purity production of type XVII collagen without the use of animal sources, laying a solid foundation for its application in skincare products and pharmaceuticals.

[0005] Patent document CN118530338A discloses a recombinant human type XVII collagen with hair follicle growth-promoting activity and its application. The recombinant human type XVII collagen is composed of a repeatedly occurring dodecapeptide amino acid sequence derived from natural human type XVII collagen. Both the dodecapeptide and the recombinant human type XVII collagen exhibit high hair follicle growth-promoting activity and can be used in cosmetics for preventing hair loss, promoting hair growth, or promoting hair follicle cell growth, such as shampoos, hair creams, hair lotions, and hair gels.

[0006] Patent document CN120248093A discloses a recombinant humanized type A collagen of type XVII and its construction and application. This protein, a humanized type XVII collagen, is composed of an intracellular region, a transmembrane region, and an extracellular region, and has biological activities such as hair follicle repair, hair regeneration, cell proliferation, and cell adhesion. It can be widely used in the fields of medicine, medical devices, biomaterials, tissue engineering, and cosmetics.

[0007] However, recombinant collagen prepared using current genetic engineering methods still suffers from problems such as poor stability, easy degradation, and low expression levels. Therefore, researching and developing a recombinant type XVII collagen with advantages such as high stability, high expression levels, and good hydrophilicity remains a challenge in current research. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a novel recombinant humanized XVII type collagen. This invention designs a recombinant humanized XVII type collagen that can be efficiently and stably expressed in Pichia pastoris strains. Furthermore, the recombinant XVII type collagen obtained by this invention can significantly upregulate the expression of VEGF and WNT10B genes, exhibiting a significant anti-hair loss effect and showing great application potential in the field of hair loss prevention.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a recombinant type XVII humanized collagen, which includes a basic repeating unit. The amino acid sequence of the basic repeating unit is formed by splicing the amino acid peptides shown in SEQ ID NO:1 and SEQ ID NO:2 sequentially from the N-terminus to the C-terminus. The number of tandem basic repeating units is 8.

[0011] Furthermore, the amino acid sequence of the basic repeating unit is shown in SEQ ID NO:3.

[0012] Furthermore, the amino acid sequence of the recombinant type XVII humanized collagen is shown in SEQ ID NO:4.

[0013] Furthermore, the present invention provides a nucleic acid molecule that encodes the aforementioned recombinant type XVII humanized collagen.

[0014] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5.

[0015] Furthermore, the present invention provides a recombinant XVII type humanized collagen recombinant vector, wherein the recombinant vector comprises the above-mentioned nucleic acid molecules.

[0016] Furthermore, the present invention provides an engineered strain comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant XVII type humanized collagen recombinant vector.

[0017] In addition, the present invention also provides the application of the recombinant type XVII humanized collagen in the preparation of pharmaceutical, cosmetic or medical device products.

[0018] Furthermore, the recombinant type XVII humanized collagen is used in the preparation of cosmetics that prevent hair loss, promote hair growth, or promote the growth of hair follicle cells.

[0019] Furthermore, the present invention provides a composition characterized in that the composition comprises the above-described recombinant type XVII humanized collagen and an acceptable excipient.

[0020] The novel recombinant humanized type XVII collagen provided by this invention is obtained by sequentially linking two fragments of human type XVII collagen, both of which are GPPs at both ends, and performing eight tandem replications. The recombinant humanized type XVII collagen prepared by this invention not only possesses advantages such as high stability, high expression levels, and good hydrophilicity, but also significantly upregulates the expression of VEGF and WNT10B genes, thus exhibiting anti-hair loss effects. Furthermore, the amino acid sequence of the recombinant humanized type XVII collagen prepared by this invention is 100% identical to the corresponding portion of natural human type XVII collagen, and its application in the human body will not induce immune rejection, demonstrating high safety and significant application value in the field of hair loss prevention.

[0021] Compared with existing technologies, the recombinant type XVII humanized collagen provided by this invention has the following advantages:

[0022] (1) The amino acid sequence of the recombinant humanized type XVII collagen provided by the present invention is 100% identical to the corresponding part of natural human type XVII collagen. It will not produce immune rejection when applied to the human body and has high safety.

[0023] (2) The recombinant XVII type humanized collagen amino acid sequence provided by this invention can be successfully expressed and secreted in a yeast expression system with high expression level and high purity. This invention uses a 50 L fermenter for fermentation, and after purification, 164.2 g of recombinant XVII type humanized collagen lyophilized sponge can be obtained with a purity of 96.0%, bacterial endotoxin <0.5 EU / mg, exogenous DNA residue <1.5 ng / mg, and host protein residue <0.05%.

[0024] (3) The recombinant XVII type humanized collagen provided by the present invention has strong solubility, and its solubility in water can reach 200 g / L.

[0025] (4) The recombinant type XVII humanized collagen provided by the present invention can significantly upregulate the expression level of the WNT10B gene. At 10 mg / mL of the recombinant type XVII humanized collagen of the present invention, the relative expression level of the WNT10B gene in human dermal papilla cells increased by 278.7% compared with the model control group.

[0026] (5) The recombinant type XVII humanized collagen provided by the present invention can significantly upregulate the expression level of VEGF gene. At 10 mg / mL of the recombinant type XVII humanized collagen of the present invention, the relative expression level of VEGF gene in human dermal papilla cells increased by 92.4% compared with the model control group. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the construction of the recombinant type XVII humanized collagen protein particle pPIC9K-hcq2.

[0028] Figure 2 SDS-PAGE electrophoresis image of recombinant type XVII humanized collagen expressed in shake flasks.

[0029] Figure 3 SDS-PAGE electrophoresis image of fermentation supernatant of recombinant type XVII humanized collagen.

[0030] Figure 4 This is an SDS-PAGE electrophoresis image of the purified recombinant type XVII humanized collagen.

[0031] Figure 5 The mass spectrum of the relative molecular mass of recombinant type XVII humanized collagen.

[0032] Figure 6 This is a graph showing the relative expression levels of the WNT10B gene.

[0033] Figure 7 This is a diagram showing the relative expression levels of the VEGF gene. Detailed Implementation

[0034] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but as long as they do not depart from the basic idea of ​​the invention, they are all within the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0035] Example 1: Design and Synthesis of Recombinant Type XVII Humanized Collagen Gene

[0036] 1. Amino acid sequence of recombinant type XVII humanized collagen:

[0037] This invention analyzes the α chain of human type XVII collagen, selects sequence fragments SEQ ID NO.1 and SEQ ID NO.2, both of which are GPP at both ends, and splices them sequentially to obtain the target sequence fragment SEQ ID NO.3 monomer. Using the target sequence fragment SEQ ID NO.3 monomer as the basic repeating unit, it is repeated 8 times in tandem to obtain the recombinant type XVII humanized collagen amino acid sequence as shown in SEQ ID NO.4.

[0038] (1) The amino acid sequence of the amino acid peptide with GPP at both ends is (SEQ ID NO:1):

[0039] GPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPP.

[0040] The amino acid sequence of the peptide with GPPs at both ends is (SEQ ID NO:2):

[0041] GPPGQKGEMGTPPGPKGDRGPAGPPGHPGPP.

[0042] (3) The amino acid of the basic repeating unit is (SEQ ID NO:3):

[0043] GPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPP.

[0044] (4) The amino acid sequence of the recombinant type XVII humanized collagen is (SEQ ID NO:4):

[0045] .

[0046] 2. Nucleotide sequence of recombinant type XVII humanized collagen:

[0047] The amino acid sequence of the above-mentioned recombinant type XVII humanized collagen was used to design corresponding nucleotide sequences based on the codon preference of Pichia pastoris. The nucleotide sequence of the recombinant type XVII humanized collagen was then optimized based on the codon preference of Pichia pastoris, resulting in the nucleotide sequence of the recombinant type XVII humanized collagen shown in SEQ ID NO. 5. The synthesis was commissioned to Nanjing GenScript Biotech Co., Ltd.

[0048] The nucleotide sequence of the recombinant type XVII humanized collagen is (SEQ ID NO:5):

[0049]

[0050] 3. Construction of recombinant plasmids:

[0051] The nucleotide sequence of the synthesized recombinant type XVII humanized collagen protein was constructed into the pPIC9K vector to obtain the recombinant type XVII humanized collagen protein particle, named pPIC9K-hcq2. A schematic diagram of the recombinant type XVII humanized collagen protein particle pPIC9K-hcq2 constructed in this invention is shown below. Figure 1 As shown.

[0052] Example 2: Preparation of recombinant XVII type humanized collagen genetically engineered strain

[0053] 1. Preparation of linearized plasmids:

[0054] The plasmid was linearized using a single enzyme digestion method. The recombinant humanized collagen protein plasmid pPIC9K-hcq2 obtained in Example 1 was extracted, digested with SalI enzyme, and then recovered by ethanol precipitation to obtain the linearized plasmid of recombinant humanized collagen protein.

[0055] 2. Preparation of competent yeast cells:

[0056] Pichia pastoris strain GS115 was inoculated into YPD liquid medium and cultured overnight at 28 ℃ and 220 rpm. The OD600 value of the bacterial culture was then measured. Initially, OD600 was 0.5, and the culture was inoculated into 50 mL of YPD liquid medium and cultured until the OD600 reached 1.3–1.5. After centrifugation at 4 ℃ and 3000 g for 5 min, the medium was discarded. The yeast cells were resuspended in 50 mL of sterile water and centrifuged at 4 ℃ and 3000 g for 5 min, and the supernatant was discarded. The yeast cells were resuspended in 25 mL of sterile water and centrifuged at 4 ℃ and 3000 g for 5 min, and the supernatant was discarded. The yeast cells were resuspended in 2 mL of pre-chilled sorbitol and centrifuged at 4 ℃ and 3000 g for 5 min, and the supernatant was discarded. Finally, the yeast cells were resuspended in 100 μL of pre-chilled sorbitol and aliquoted into 80 μL tubes.

[0057] 3. Pichia pastoris transformation:

[0058] Take 10 μL of the prepared recombinant type XVII humanized collagen linearized plasmid and add it to Pichia pastoris competent cells. Mix well and transfer to an electroporation cuvette that has been pre-chilled on ice. Incubate on ice for at least 5 min. Wipe the outer wall of the electroporation cuvette dry and place it in an electroporator. Add 1 mL of sorbitol to suspend the bacterial cells. Then transfer the liquid to a 1.5 mL centrifuge tube and incubate at 28 ℃ for 3–12 h. Spread the mixture on MD plates and incubate at 28 ℃ until colonies grow.

[0059] 4. Validation of expression of recombinant type XVII humanized collagen in genetically engineered strains:

[0060] Single colonies were picked and cultured in 10 mL of BMGY medium at 28 °C and 220 rpm until OD600 = 2–6. The cells were centrifuged at 3000 g for 5 min, and resuspended in 10 mL of BMGY medium to bring the OD600 to 1. The cells were then cultured at 28 °C and 220 rpm, with methanol added every 24 hours until a final concentration of 0.5% was reached. After induction for 72 h, the supernatant was collected by centrifugation and analyzed by SDS-PAGE.

[0061] The results of SDS-PAGE electrophoresis analysis of the recombinant type XVII humanized collagen expressed in shake flasks by this invention are as follows: Figure 2 As shown. Figure 2 This is an SDS-PAGE electrophoresis image of recombinant humanized collagen XVII expressed in shake flasks (M: Marker; 1: Negative control; 2-3: Verification of recombinant humanized collagen expression in Pichia pastoris genetically engineered strains). From... Figure 2 It is known that the novel recombinant type XVII humanized collagen amino acid sequence provided by this invention was successfully expressed and secreted in the Pichia pastoris expression system.

[0062] Example 3: Preparation of recombinant type XVII humanized collagen

[0063] 1. Fermentation of recombinant XVII type humanized collagen:

[0064] The recombinant XVII type humanized collagen expression strain obtained in Example 2 was inoculated into YPD medium and cultured at 28°C and 220 rpm until the seed culture concentration OD600 reached 15-25, and no contaminants were observed under microscopic examination. The seed culture was then inoculated into a 50 L fermenter at a 5% inoculation rate. Initial culture conditions were: agitator speed of 100 rpm, fermenter pressure of 0.05 MPa, pH controlled at 5.2, and temperature at 30°C. Dissolved oxygen was maintained above 30% by adjusting the air flow rate and agitation speed.

[0065] When the carbon source in the initial fermentation medium was depleted, dissolved oxygen rose sharply, and 50% glycerol was added to replenish the carbon source. Once the carbon source (50% glycerol) was exhausted, glycerol feeding was stopped, and methanol was added to initiate induction. Dissolved oxygen (DO) was maintained above 5% by adjusting the turbine rotation speed, air flow rate, and methanol flow rate. Fermentation supernatants were collected at 23 h, 47 h, 68 h, and 88 h for SDS-PAGE electrophoresis analysis.

[0066] The SDS-PAGE electrophoresis image of the fermentation supernatant of recombinant type XVII humanized collagen obtained by this invention is shown below. Figure 3 As shown. Figure 3 SDS-PAGE electrophoresis images of the fermentation supernatant of recombinant type XVII humanized collagen (M: Marker; 1-5: SDS-PAGE electrophoresis images after 23 h, 47 h, 68 h, and 88 h induction, and after 5-fold dilution of the sample). Figure 3 It can be seen that after 88 hours of induction, the supernatant of fermentation diluted 5 times had an electrophoresis band brightness that was about 10 times that of the 66.2 KD standard.

[0067] 2. Purification of recombinant type XVII humanized collagen:

[0068] Fermentation was terminated after 88 h of induction, and the fermentation product was obtained. The fermentation product was centrifuged at 10 ℃ and 8000 rpm for 20 min to obtain the fermentation filtrate. The fermentation filtrate was used to capture proteins through a cation exchange medium and eluted with elution buffer (10 mmol / L PB buffer, pH 8.0) to obtain recombinant humanized collagen type XVII. The purified recombinant humanized collagen type XVII was detected by SDS-PAGE electrophoresis.

[0069] The SDS-PAGE electrophoresis results of the purified recombinant type XVII humanized collagen of this invention are as follows: Figure 4 As shown. Figure 4 The image shows the SDS-PAGE electrophoresis results of the purified recombinant humanized collagen type XVII (1: recombinant humanized collagen type XVII; M: marker).

[0070] from Figure 4 It is evident that the recombinant type XVII humanized collagen obtained by this invention has a single band and high purity, with a purity of 96.0%. This invention uses a 50 L fermenter for fermentation, and after purification, 164.2 g of lyophilized sponge containing recombinant type XVII humanized collagen can be obtained, with bacterial endotoxin <0.5 EU / mg, exogenous DNA residue <1.5 ng / mg, and host protein residue <0.05%.

[0071] Example 4: Determination of molecular weight of recombinant type XVII humanized collagen

[0072] 1. Experimental Method:

[0073] The recombinant type XVII humanized collagen prepared in this invention has a theoretical molecular weight of 53051.78 Da, and the apparent molecular weight detected by SDS-PAGE is approximately 66.2 KD, which is larger than the theoretical molecular weight. The relative molecular mass of the recombinant type XVII humanized collagen prepared in Example 3 was analyzed by Shanghai Aipudikang Biotechnology Co., Ltd. using a high-resolution Xevo G2-XS QTof (Waters Corporation) mass spectrometer.

[0074] 2. Experimental Results:

[0075] Experimental results are as follows Figure 5 As shown.

[0076] Figure 5 This is the relative molecular mass mass mass spectrum of recombinant humanized collagen type XVII. Figure 5 It is known that the molecular weight of the recombinant type XVII humanized collagen obtained in Example 3 of this invention is 53050.9 Da, which is consistent with the theoretical value. The apparent molecular weight of the recombinant type XVII humanized collagen is greater than the theoretical value, which may be because there are many hydrophilic residues in the collagen, resulting in a weaker binding ability between the protein and SDS, a reduced negative charge load on the target protein, and therefore a slower migration rate and a shorter migration distance.

[0077] Example 5: Solubility determination of recombinant type XVII humanized collagen

[0078] 1. Experimental Method:

[0079] The recombinant humanized collagen type XVII prepared in Example 3 was dissolved in water, with shaking for 30 seconds every 5 minutes. The time required for dissolution and the appearance of the solution after dissolution were observed. The solution was considered soluble if no solute particles or droplets were visible to the naked eye within 30 minutes.

[0080] Experimental results:

[0081] 2. The experimental results are shown in Table 1.

[0082] Table 1. Solubility results of recombinant humanized collagen type XVII

[0083] Protein concentration (g / L) Dissolution time Solubility 1 2 min Soluble, dissolves to form a colorless, clear solution. 5 2 min Soluble, dissolves to form a colorless, clear solution. 10 2 min Soluble, dissolves to form a colorless, clear solution. 50 5 min Soluble, dissolves to form a colorless, clear solution. 100 20 min Soluble, dissolving into a pale yellow, slightly viscous, clear solution. 140 25 min Soluble, dissolving to form a pale yellow, slightly viscous, clear solution. 160 25 min Soluble, dissolving to form a pale yellow, slightly viscous, clear solution. 200 30 min Soluble, dissolving into a pale yellow, viscous, clear solution.

[0084] As shown in Table 1, the recombinant type XVII humanized collagen prepared by this invention has strong solubility, with a solubility of up to 200 g / L in water.

[0085] Example 6: Cytotoxicity detection of recombinant type XVII humanized collagen

[0086] Experimental methods:

[0087] The recombinant humanized collagen of type XVII prepared in Example 3 was subjected to cytotoxicity testing.

[0088] (1) Collect human dermal papilla cells with a confluence of 80% to 90% in T75 bottles, count them, adjust the cell density and seed them in 96-well plates, and incubate them in a carbon dioxide incubator for 24 h.

[0089] (2) Remove the culture plate, discard the culture medium in the wells, add working solutions of different concentrations respectively, and continue to incubate in a carbon dioxide incubator for 24 h;

[0090] (3) After taking out the 96-well plate and taking pictures, incubate it in CCK8 working solution in the dark for 1-2 hours.

[0091] (4) The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0092] (5) Calculation of cell survival rate results.

[0093] Survival rate (%) = OD450e / OD450b × 100% (where OD450e is the absorbance of the test sample group and the positive control group (PC); OD450b is the absorbance of the blank control group (NC).

[0094] 2. Experimental Results:

[0095] The experimental results are shown in Table 2.

[0096] Table 2. Cytotoxicity Results of Recombinant Type XVII Humanized Collagen

[0097]

[0098] As shown in Table 2, the recombinant type XVII humanized collagen prepared by this invention has good safety. Subsequent tests were conducted using concentrations of 10 mg / mL, 1.25 mg / mL, and 0.1563 mg / mL.

[0099] Example 7: Evaluation of hair loss prevention by recombinant type XVII humanized collagen

[0100] 1. Experimental Method:

[0101] The regulatory effects of the recombinant type XVII humanized collagen prepared in Example 3 on the WNT10B and VEGF genes were investigated.

[0102] (1) Collect human dermal papilla cells with a confluence of 80% to 90% in T75 bottles, count them, adjust the cell density, seed them into culture plates, and incubate them in a carbon dioxide incubator for 24 h.

[0103] (2) Remove the culture plate, discard the culture medium in the wells, and add working solutions of different concentrations. NC is the blank control group; M is the model control group, with dihydrotestosterone added; PC is the positive control group, with dihydrotestosterone and quercetin added; S1 is the experimental group, with dihydrotestosterone and 10 mg / mL of the recombinant XVII type humanized collagen of the present invention added; S2 is the experimental group, with dihydrotestosterone and 1.25 mg / mL of the recombinant XVII type humanized collagen of the present invention added; S3 is the experimental group, with dihydrotestosterone and 0.1563 mg / mL of the recombinant XVII type humanized collagen of the present invention added. Continue to culture in a carbon dioxide incubator for 24 h.

[0104] (3) Remove the culture plate, discard the culture medium in the wells, add 1 mL of PBS to each well to rinse and discard, repeat once, aspirate the liquid in the wells, place on ice for later use, or use trypsin digestion treatment, collect the cells and store them at -80 ℃.

[0105] (4) Extract RNA according to the instructions of the total RNA extraction kit for cultured cells / bacteria.

[0106] (5) Perform reverse transcription to obtain cDNA according to the instructions of HiScript® II 1st Strand cDNA Synthesis Kit (+gDNAwiper).

[0107] (6) The expression of relevant genes in cells was detected by real-time PCR.

[0108] (7) Using GAPDH as an internal reference, qPCR was used to detect and calculate the relative expression levels of WNT10B and VEGF in each group of cDNA samples (2). -∆∆Cq ).

[0109] (8) Statistical analysis: The relative expression levels (Mean±SD) of the sample group and the model group were statistically analyzed (t-test) and plotted.

[0110] 2. Experimental Results:

[0111] 2.1 The regulatory effects of recombinant type XVII humanized collagen on the WNT10B gene are as follows: Figure 6 As shown in Table 3.

[0112] Figure 6The graph shows the relative expression levels of the WNT10B gene, where: NC is the blank control group; M is the model control group, treated with dihydrotestosterone (DHT); PC is the positive control group, treated with DHT + quercetin; S1 is the experimental group, treated with DHT + 10 mg / mL recombinant humanized type XVII collagen of this invention; S2 is the experimental group, treated with DHT + 1.25 mg / mL recombinant humanized type XVII collagen of this invention; S3 is the experimental group, treated with DHT + 0.1563 mg / mL recombinant humanized type XVII collagen of this invention; * indicates a statistically significant difference compared with group M (p<0.05); ** indicates a significant statistical difference compared with group M (p<0.01); *** indicates an extremely significant statistical difference compared with group M (p<0.001).

[0113] Table 3. Relative expression levels of WNT10B (normalized to M)

[0114] Sample <![CDATA[2 -∆∆CT ]]> SD growth rate P-value (compared to M) NC 1.402 0.091 40.2% 0.0080 M 1.000 0.109 / / PC 2.221 0.251 122.1% 0.0015 S1 3.787 0.274 278.7% <0.0001 S2 1.859 0.322 85.9% 0.0197 S3 1.115 0.150 11.5% 0.3432

[0115] Depend on Figure 6 As shown in Table 3, the relative expression levels of the WNT10B gene in cells prepared by the recombinant type XVII humanized collagen of the present invention increased by approximately 278.7%, 85.9%, and 11.5% compared with the model control group (M) at concentrations of 10 mg / mL, 1.25 mg / mL, and 0.1563 mg / mL, respectively. Among them, the expression levels at concentrations of 10 mg / mL and 1.25 mg / mL were significantly upregulated compared with the model control group (M) (p<0.05).

[0116] In conclusion, under the conditions of this experiment, recombinant humanized type XVII collagen at concentrations of 10 mg / mL and 1.25 mg / mL significantly upregulated WNT10B gene expression, indicating that the samples have an anti-hair loss effect.

[0117] 2.2 The regulatory results of recombinant type XVII humanized collagen on the VEGF gene are as follows: Figure 7 As shown in Table 4.

[0118] Figure 7The graph shows the relative expression levels of the VEGF gene. NC represents the blank control group; M represents the model control group, treated with dihydrotestosterone (DHT); PC represents the positive control group, treated with DHT + quercetin; S1 represents the experimental group, treated with DHT + 10 mg / mL recombinant humanized type XVII collagen of this invention; S2 represents the experimental group, treated with DHT + 1.25 mg / mL recombinant humanized type XVII collagen of this invention; S3 represents the experimental group, treated with DHT + 0.1563 mg / mL recombinant humanized type XVII collagen of this invention. * indicates a statistically significant difference compared to group M (p<0.05); ** indicates a significant statistical difference compared to group M (p<0.01); *** indicates an extremely significant statistical difference compared to group M (p<0.001).

[0119] Table 4. Relative VEGF expression levels (normalized to M)

[0120] Sample <![CDATA[2 -∆∆CT ]]> SD growth rate P-value (compared to M) NC 1.881 0.270 88.1% 0.009 M 1.000 0.174 / / PC 3.778 0.335 277.8% 0.0002 S1 1.924 0.195 92.4% 0.0036 S2 1.571 0.256 57.1% 0.0331 S3 1.089 0.225 8.9% 0.6166

[0121] Depend on Figure 7 As shown in Table 4, the relative expression levels of the VEGF gene in cells prepared by the recombinant type XVII humanized collagen at concentrations of 10 mg / mL, 1.25 mg / mL, and 0.1563 mg / mL increased by approximately 92.4%, 57.1%, and 8.9% respectively compared with the model control group (M). Among them, the expression levels at concentrations of 10 mg / mL and 1.25 mg / mL were significantly upregulated compared with the model control group (M) (p<0.05).

[0122] In conclusion, under the conditions of this experiment, recombinant humanized type XVII collagen at concentrations of 10 mg / mL and 1.25 mg / mL significantly upregulated VEGF gene expression, indicating that the samples have an anti-hair loss effect.

[0123] The above are merely further embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope disclosed in the present invention, based on the technical solution and concept of the present invention, shall fall within the scope of protection of the present invention.

Claims

1. A recombinant type XVII humanized collagen, characterized in that, The amino acid sequence of the recombinant type XVII humanized collagen is shown in SEQ ID NO:

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant type XVII humanized collagen as described in claim 1.

3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

4. A recombinant XVII type humanized collagen recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule as described in claim 2 or claim 3.

5. An engineered bacterial strain, characterized in that, The engineered strain comprises the nucleic acid molecule of claim 2 or claim 3, or the recombinant humanized collagen recombinant vector of claim 4.

6. A composition, characterized in that, The composition comprises the recombinant type XVII humanized collagen of claim 1 and an acceptable excipient.

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