Multi-effect repair type collagen and application thereof

Abstract

The invention discloses a multi-effect repair type collagen and application thereof, and belongs to the technical field of synthetic biology. Transdermal peptide TD-1, a transmembrane region of human XVII type collagen, an extracellular sixteenth non-collagen region, an extracellular fifteenth collagen region and a coding gene of a His tag are selected to be connected in series, and a gene sequence is optimized through pichia pastoris codon selection preference; and then the T-COL17R3 is constructed and expressed to obtain the T-COL17R3. Efficacy experiments prove that compared with similar products sold in the market, T-COL17R3 not only has excellent transdermal performance, but also has better cell proliferation and migration promoting capability, better capability of resisting early saccharification product ketoamine and later saccharification product dicarbonyl compounds, better capability of scavenging free radicals and resisting elastase, and can be used for preparing the anti-epidermal drug. In addition, synthesis of melanin in B16-F10 cells can be reduced, so that T-COL17R3 has excellent application potential in development of pharmaceutical compositions or skin care products with repairing, anti-wrinkle and firming or whitening and brightening effects.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a multi-effect repair collagen and applications thereof. Background Art

[0002] The skin is the body's largest organ, protecting us from mechanical stress and pathogens. To achieve these functions, basal keratinocytes must continuously detach from the basement membrane (BM), divide, and migrate upward. The BM, also known as the dermal-epidermal junction, anchors the epidermis to the dermis through various extracellular matrix proteins beneath the dermis. It performs functions such as transmitting signals and nutrients and maintaining skin's structural firmness. A healthy BM performs multiple biological functions, ensuring smooth circulation between the epidermis, BM, and dermis, and maintaining healthy and intact skin. Type XVII collagen is the core element for the stability of the basement membrane zone. It combines with integrins to form hemidesmosomes, which connect type IV and type VII collagen to form a complete, supportive semi-permeable membrane structure that stabilizes the epidermal connection and maintains skin integrity. In photoaged skin, the basement membrane flattens, the expression of type XVII collagen decreases significantly, the hemidesmosomes become loose, and the skin structure and function become abnormal, manifesting as skin fragility and dysfunction. Supplementation of type XVII collagen can repair the basement membrane zone, regulate cell self-purification, and promote collagen regeneration. In the process of skin wound repair, type XVII collagen plays an important role by affecting stem cell migration, proliferation, and differentiation. With the development of synthetic biology technology, recombinant type XVII collagen can be obtained as a new type of therapeutic collagen and anti-aging raw material, bringing new breakthroughs to skin anti-aging.

[0003] Compared to animal-derived collagen, recombinant humanized collagen not only exhibits superior bioactivity, biocompatibility, and water solubility, but also carries a lower pathogen risk. Type XVII collagen is scarce, and animal extraction is not feasible. However, the large-scale application of genetic engineering technology has successfully overcome the bottleneck of large-scale production of type XVII collagen through the recombinant expression of exogenous proteins.

[0004] A prior application (Application No. 202510847175.4) proposed a highly transdermal recombinant humanized type XVII collagen, its preparation method, and application. By selecting the gene encoding the transdermal peptide TD-1, optimized for Pichia pastoris codon usage, and the genes encoding different functional fragments of human type XVII collagen, along with a His tag, and sequentially expressing them in tandem, two recombinant humanized type XVII collagens, named T-COL17-NC16 and T-COL17, were constructed. Efficacy experiments demonstrated that these two recombinant humanized type XVII collagens exhibited superior transdermal permeability, in addition to superior repair, anti-wrinkle, firming, and whitening and brightening effects, compared to similar commercially available products. To further enhance the application of recombinant humanized type XVII collagen in pharmaceutical compositions or skin care products, the present invention proposes a multi-functional repair collagen and its application. Summary of the Invention

[0005] The purpose of the present invention is to provide a multi-effect repair collagen and its application in order to solve the above problems.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions: As a first aspect of the present invention, a multifunctional repair collagen is disclosed. The multifunctional repair collagen T-COL17R3 comprises a transdermal peptide TD-1, a partial functional fragment of type XVII collagen, and a His tag. The partial functional fragment of type XVII collagen is selected from the transmembrane region, the extracellular sixteenth non-collagen region, and the extracellular fifteenth collagen region of type XVII collagen. The amino acid sequence of the multifunctional repair collagen T-COL17R3 is shown in SEQ ID NO.1.

[0007] As a second aspect of the present invention, a polynucleotide is also disclosed, encoding the multifunctional repair collagen described above. The nucleotide sequence of the multifunctional repair collagen T-COL17R3 is shown in SEQ ID NO.3.

[0008] As a third aspect of the present invention, a recombinant plasmid is also disclosed. The recombinant plasmid comprises the polynucleotide sequence described above and can correspondingly translate and express the multi-functional repair collagen T-COL17R3 described above.

[0009] As a fourth aspect of the present invention, a host cell is also disclosed. The host cell comprises the recombinant plasmid as described above or expresses the multifunctional repair collagen T-COL17R3 as described above.

[0010] As a further optimized solution of the present invention, the host cell is Pichia pastoris.

[0011] As a fifth aspect of the present invention, a method for preparing the multi-functional repair collagen as described above is also disclosed, comprising the following steps: (1) inoculating any of the host cells described above into a fermentation medium for fermentation culture, and after the fermentation culture is completed, centrifuging and collecting the supernatant to obtain a fermentation broth; (2) The fermentation liquid obtained in step (1) is separated and purified, and the protein is detected to obtain the multi-functional repair collagen.

[0012] As a sixth aspect of the present invention, also disclosed is a use of any of the multi-effect repair collagen described above in the preparation of a pharmaceutical composition or a skin care product.

[0013] As a further optimization solution of the present invention, the pharmaceutical composition or skin care product has at least one of the effects of repairing, anti-wrinkle and firming, or whitening and brightening.

[0014] The beneficial effects of the present invention are: The present invention concatenates the genes encoding the transdermal peptide TD-1, the transmembrane region of human type XVII collagen, the extracellular non-collagenous region 16, the extracellular collagen region 15, and a His tag. The gene sequence is optimized using Pichia pastoris codon usage preferences. The recombinant humanized type XVII collagen, T-COL17R3, is then constructed and expressed. Furthermore, efficacy experiments have demonstrated that T-COL17R3 exhibits multiple benefits, including repair, anti-wrinkle firming, and whitening and brightening, compared to similar commercially available products. Its excellent transdermal ability suggests promising applications in pharmaceutical compositions or skincare products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a test of the ability of recombinant humanized type XVII collagen to promote cell proliferation; Figure 2 This is the test result of the ability of recombinant humanized type XVII collagen to promote cell migration; Figure 3 This is a graph showing changes in cell scratch area at different times; Figure 4 This is the result of the cytotoxicity test of recombinant humanized type XVII collagen on 3T3 cells; Figure 5 This is the result of the test on the adhesion promoting effect of recombinant humanized type XVII collagen on 3T3 cells; Figure 6 This is the result of the test on the inhibition rate of recombinant humanized type XVII collagen on melanin production in B16-F10 cells; Figure 7 is a schematic diagram of the diffusion cell used in the transdermal performance test; Figure 8This is the 24-hour transdermal performance test result of recombinant humanized type XVII collagen and similar products on the market; Figure 9 This is the 48-hour transdermal performance test result of recombinant humanized type XVII collagen and similar products on the market; Figure 10 It is the result of the test of aminoguanidine (AG) anti-non-enzymatic glycation ability; Figure 11 This is the result of the test of the ability of recombinant humanized type XVII collagen to resist ketoamine, an early glycation product; Figure 12 This is the result of the test on the ability of recombinant humanized type XVII collagen to resist dicarbonyl compounds, a product of advanced glycation; Figure 13 This is the result of the test on the anti-glycosylation ability of recombinant humanized type XVII collagen in HACAT cells; Figure 14 This is the result of the ABTS free radical scavenging ability test of recombinant humanized type XVII collagen; Figure 15 This is the result of the positive control ABTS free radical scavenging ability test. DETAILED DESCRIPTION

[0016] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0017] Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art. If specific conditions are not specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0018] YPD liquid medium: 10g yeast extract, 20g tryptone, 20g glycerol, dilute to 1000ml with ultrapure water, and sterilize at 121℃ for 20min; YPD solid medium: Add 15 g agar per 1000 ml of YPD liquid medium; BMGY medium: 10 g yeast extract, 20 g trypton, 40 g glycerol, dilute to 800 ml, add 100 ml 1 M phosphate buffer, sterilize at 121°C for 20 min, cool, add 100 ml filter-sterilized 10× YNB and 2 ml biotin.

[0019] 0.2M phosphate buffer (PB): Dissolve 27.22g K2HPO4 and 8g NaCl in 800ml of ultrapure water, adjust the pH to 7.4, and make up to 1000ml. This is solution A.

[0020] 3M NaCl: Dissolve 175.32 g NaCl in 1000 ml ultrapure water. This is Solution B.

[0021] Nickel column chromatography Binding Buffer: 100 ml of solution A, 100 ml of solution B, add ultrapure water to 800 ml, adjust the pH to 7.4, and make up to 1000 ml.

[0022] Nickel column chromatography Wash Buffer: 100 ml of solution A, 100 ml of solution B, add ultrapure water to 800 ml, adjust the pH to 7.4, and make up to 1000 ml.

[0023] Nickel column chromatography Elution Buffer: 100 ml of solution A, 100 ml of solution B, 34 g of imidazole, add ultrapure water to 800 ml, adjust the pH to 7.4, and make up to 1000 ml.

[0024] 0.02M phosphate buffer (PB): Dissolve 2.4g NaH2PO4 and 2.8g Na2HPO4 in 800ml ultrapure water, adjust the pH to 7.4, and make up to 1000ml.

[0025] 1. Gene design and synthesis 1.1 Design and synthesis of the gene encoding T-COL17R3 The transdermal peptide TD-1, the transmembrane region of type XVII collagen, the extracellular sixteenth non-collagenous region, and the largest extracellular fifteenth collagen region were selected and concatenated with the coding gene of the His tag. The gene sequence was optimized according to the codon usage preference of Pichia pastoris to obtain the target gene sequence. Then, the optimized gene full sequence was obtained through whole gene synthesis (synthesized by Jinweizhi Company). The full gene sequence is shown in SEQ ID NO.3, and the encoded collagen protein is T-COL17R3, and its amino acid sequence is shown in SEQ ID NO.1.

[0026] 1.2. Construction of recombinant expression engineering bacteria The plasmid and the constitutive vector plasmid pGAPZα were double-digested, and the target fragment and the plasmid pGAPZα expression vector fragment were recovered by gel excision. The target fragment and the expression vector were ligated using ligase, and the ligation product was transformed into competent E. coli TOP10. Positive clones were screened on LB resistance plates containing Zeocin to successfully obtain the recombinant constitutive expression plasmid.

[0027] The recombinant constitutive expression plasmid was linearized and electroporated into competent Pichia pastoris X33. The glyceraldehyde 3-phosphate dehydrogenase promoter in X33 eliminates the need for methanol during fermentation, avoiding the contamination and risks associated with the use of large amounts of methanol during production, making it more suitable for large-scale production. Dot-blot and Western-blot analysis using Zeocin as a resistance marker and a his-tag antibody were performed to obtain the recombinant Pichia pastoris.

[0028] 1.3 Fermentation Expression Streak the engineered recombinant Pichia pastoris onto a YPD solid medium plate and incubate at 30°C until a single colony forms. A single colony is selected and inoculated into 10 ml of YPD liquid medium and cultured overnight at 30°C with shaking at 220 rpm to create the primary seed solution. Add 1 ml of the primary seed solution to 200 ml of BMGY medium and culture at 30°C with shaking at 220 rpm for 24 hours to create the secondary seed solution. This secondary seed solution is then added to 4 L of BMGY liquid medium and cultured at 30°C with shaking at 220 rpm for 60 hours. The supernatant is then centrifuged and used for subsequent isolation and purification experiments.

[0029] 1.4 Separation and purification T-COL17-R3 is expressed intracellularly due to the presence of a transmembrane region, and its intracellular protein was extracted using snailase for subsequent separation and purification experiments.

[0030] 1.4.1. Extraction of intracellular proteins by snail enzyme lysis The fermentation broth was centrifuged at 6000 rpm for 5 minutes, and the cells were harvested and weighed. Snail enzyme was dissolved in SE buffer to a concentration of 40 mg / ml. For each gram of cells, 5 ml of isotonic sorbitol solution (pH 5.8-7.2), 1 ml of snail enzyme, and 20 μl of β-mercaptoethanol were added. Enzymatic digestion was performed at 37°C for 2 hours. Centrifuge at 8000 rpm for 5 minutes, discard the supernatant, and collect the precipitate. Resuspend the cells in an appropriate amount of sorbitol, centrifuge again at 8000 rpm for 10 minutes, discard the supernatant, collect the precipitate, and repeat the wash process. Resuspend the cells in an appropriate amount of ddH2O, freeze at -80°C for 30 minutes, and then bring to room temperature for dissolution. Repeat this process three times. The lysed solution was centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected as the yeast protein solution for subsequent experimental analysis.

[0031] 1.4.2. Obtaining supernatant samples The obtained supernatants were filtered through 0.22 μm membranes to obtain flow-throughs, which were adjusted to pH 7.4 with aqueous ammonia and used as supernatant samples.

[0032] 1.4.3 Nickel column affinity chromatography Rinse the nickel column with 5-10 column volumes of ultrapure water to flush out the 20% ethanol in the nickel column (stop when the UV value remains unchanged or fluctuates steadily around a certain value); add 10 column volumes of Binding Buffer to equilibrate the column (the Binding Buffer and the supernatant sample should be placed on ice to minimize protein degradation loss); add the supernatant sample to the column, control the flow rate to 5 ml / min, and collect the flow-through; then add 10 column volumes of Wash Buffer to wash the column (to elute impurities) to obtain a wash solution; then elute the protein with Elution Buffer to obtain an eluate. Start collecting when the UV280 value rises slightly, and stop collecting when the value decreases slowly to obtain a crude protein extract.

[0033] 1.4.4 Desalting column desalting The crude protein extract was loaded onto a desalting column using PB as the washing buffer. Collection began when UV280 increased and stopped when the conductivity increased to obtain recombinant humanized type XVII collagen T-COL17R3. The protein concentration was detected by BCA method, and the purity of the collected protein was detected by SDS-PAGE.

[0034] 2. Efficacy test To further illustrate the efficacy of T-COL17R3 synthesized in the present invention, in the following efficacy tests, a similar product, commercially available recombinant humanized type XVII collagen, and T-COL17-NC16 disclosed in a prior application (application number 202510847175.4) were used as controls.

[0035] The commercially available recombinant humanized type XVII collagen was prepared in the form of the above-mentioned genetic engineering recombinant expression of exogenous protein, and its amino acid sequence was obtained by repeating the sequence shown in SEQ ID NO. 2 three times.

[0036] 2.1. Repair efficacy test 2.1.1. Detection of cell proliferation ability A 96-well plate was prepared by adding the prepared HACAT cell suspension (purchased from Nanjing Kebai Biotechnology Co., Ltd.) to a density of 10,000 cells per well. The edge wells were filled with sterile PBS. The seeded cell culture plate was cultured in an incubator for approximately 24 hours, until the cells were fully attached and growing stably. The plate was then removed, the cell culture medium discarded, and 100 µl of a gradient of drug concentrations was added. Six replicates were set up. The plates were incubated at 37°C in 5% CO2 for 24 hours or for an appropriate time (calculated based on the time of drug administration). The drug effects were observed under an inverted microscope. The culture medium was discarded, and each well was washed twice with sterile PBS. 100 µl of culture medium and 10 µl of CCK-8 were added to each well. The plates were incubated at 37°C for approximately 1 hour. The absorbance at 450 nm was measured using a microplate reader. The relative cell proliferation rate of each experimental group was calculated using the value of the negative control group as a baseline.

[0037] Relative cell proliferation rate = (OD 实验组 / OD 对照组 )×100%.

[0038] The results are as follows Figure 1 As shown, T-COL17R3 has a good ability to promote cell proliferation, which is comparable to the effect of T-COL17-NC16. At a concentration of 40 μg / ml, the ability of T-COL17R3 to promote cell proliferation is nearly 1.2 times that of the negative control group.

[0039] 2.1.2. Cell migration-promoting ability assay HACAT cells were seeded into 12-well plates at a density of 200,000 cells / well and cultured in a humidified incubator with DMEM supplemented with 10% FBS until confluent. Using a sterile 1ml pipette tip, scratch the confluent 12-well plate (scratch pattern). Remove detached cells with PBS and add fresh culture medium and drug at a rate of 1ml per well. Observe and photograph cells at 0, 6, 10, and 24 hours after scratching. Cell migration rate was calculated using the following formula.

[0040] Cell migration rate = {(initial scratch area - scratch area at time t) / initial scratch area} × 100%.

[0041] The results are shown in Table 1. Figure 2 and Figure 3 shown.

[0042] Table 1 Results of the test on the ability of recombinant humanized type XVII collagen to promote cell migration ; The results showed that T-COL17R3 had a significant promoting migration effect on cells at a concentration of 5μg / ml after acting on them for 24 hours, with the average cell migration rate reaching 100%, which was significantly better than T-COL17-NC16 and similar products on the market.

[0043] 2.1.3. Adhesion-promoting effect on 3T3 cells (1) Toxicity test on 3T3 cells When the adherent cells grow to about 80%-90% confluence, remove the culture supernatant, trypsinize the cells according to the cell passage method to obtain a single cell suspension, aspirate 50 μL of the cell suspension for cell counting, and adjust the cell density to 1×10 5 The experiment was conducted in a 96-well culture plate, with 100 μL of cell suspension in each well. Six parallel wells were set up, and sterile PBS was added to the edge wells to prevent evaporation. The inoculated cell culture plate was placed in an incubator for 24 hours. The culture supernatant was removed, and 100 μL of the test drug at various concentrations was added. The cells were incubated in the cell culture incubator for another 24 hours or for an appropriate time, calculated based on the dosing time and adjusted according to experimental needs. Cell morphology was observed under a microscope, and the cells were washed twice with PBS. 100 μL of DMEM medium and 10 μL of CCK8 reagent were added to each well. The cells were incubated at 37°C for approximately 60 minutes. The absorbance at 450 nm was measured with a microplate reader. The relative cell proliferation rate was calculated based on the absorbance.

[0044] Relative cell proliferation rate = (OD 实验组 / OD 对照组 )×100%.

[0045] The results are as follows Figure 4 As shown in the results, a concentration of 10 μg / ml had no obvious cytotoxicity to 3T3 cells and this concentration could be used in subsequent adhesion promotion experiments.

[0046] (2) 3T3 cell adhesion test Dermal fibroblasts can secrete extracellular matrix components such as collagen and elastin to maintain the mechanical properties of the skin. The reduction of their adhesion will lead to the downregulation of type I and type III collagen gene expression, causing the collapse of the extracellular matrix structure, gradual sagging of the skin, and deepening of wrinkles. In addition, the migration ability of fibroblasts with weakened adhesion is also gradually reduced, leading to delayed wound healing. This phenomenon is more common in elderly skin.

[0047] The present invention uses the NIH / 3T3 mouse fibroblast model to systematically evaluate the cytotoxicity and adhesion-promoting activity of recombinant human type XVII collagen on fibroblasts. The specific steps are as follows: The sample was diluted to 10 μg / mL artificial basement membrane glue using DMEM medium without fetal bovine serum. After mixing, 100 μL was added to each well of a 96-well plate and incubated in a 4°C refrigerator overnight. Remove the coating solution and wash the plate three times with serum-free medium; Single cell suspension was prepared by cell passage, and cells were collected by centrifugation at 300g for 3 minutes. Cells were resuspended in serum-free DMEM medium and the cell density was adjusted to 5×10 5 / mL; 100 μL was inoculated into each well of a 96-well plate, and experimental group, control group, blank group and standard group were set up respectively; Experimental group: collagen basement membrane + cells to be tested; Control group: collagen basement membrane + serum-free culture medium; Blank group: serum-free culture medium containing cells to be tested; Standard group: serum-free medium; Six replicate wells were set up in the experimental group, and three replicate wells were set up in each of the other groups. The edge wells were filled with sterile PBS buffer; Incubate the cells at 37°C and 5% CO2 for 2 hours (the incubation time can be adjusted appropriately according to the cell status and experimental requirements); The supernatant was removed (no operation was performed for the blank and standard groups), and the cells were washed three times with PBS. 100 μL of fresh culture medium and 10 μL of CCK-8 detection solution were added to each well (10 μL of CCK-8 detection solution was directly added to the blank and standard groups; The absorbance of each sample well at a wavelength of 450 nm was measured by a microplate reader, and the test data was recorded and saved; Calculate the cell adhesion rate: cell adhesion rate = [(cell OD of experimental group - OD of control group) / (cell OD of blank group - OD of standard group)] × 100% All operations must be performed under sterile conditions; avoid generating bubbles during the coating process; and thoroughly mix the cell suspension during cell inoculation.

[0048] The results are as follows Figure 5 As shown in the cell adhesion assay, T-COL17R3, which contains a transmembrane segment, exhibited superior adhesion-promoting properties, outperforming T-COL17-NC16 and similar commercial products. T-COL17R3 promotes cell attachment and anchoring to the ECM, providing favorable conditions for the development of the extracellular microenvironment.

[0049] 2.2 Whitening and brightening efficacy test 2.2.1. Effect of inhibiting melanin activity Mouse melanoma B16 F10 cells (purchased from ATCC, model CRL-6475) synthesize and secrete melanin, an amino acid derivative that reacts with sodium hydroxide to produce a water-soluble compound. The melanin content was calculated by measuring UV absorbance at 405 nm. Melanocyte stimulating hormone (αMSH) was used to treat mouse melanoma B16 F10 cells. The difference between the melanin secreted by the cells and the sample group was calculated to determine the inhibition of melanin production and evaluate the whitening activity.

[0050] The detection method is as follows: First, the effect of recombinant humanized type XVII collagen on the proliferation of melanoma cells B16-F10 was tested, and the concentration that had no effect on the proliferation of B16-F10 cells was selected for subsequent experiments; Take a 6-well plate and add 2 ml of B16-F10 cell suspension to each well to make the cell density 4×10 4 cells / well, and then placed in an incubator containing 5% CO2 for 24 hours, and the supernatant culture medium was removed and discarded; according to the sample addition information in Table 5, 2 ml of solution was added to each well, and they were recorded as blank control group, model group, positive control group and experimental group respectively; placed in an incubator containing 5% CO2, and continued to incubate at 37℃ for 72 hours (timed by the administration time), and the supernatant culture medium was discarded; then re-sampled according to Table 2, and then 2 ml of DMEM complete medium was added to each well, and incubated in an incubator containing 5% CO2 for 72 hours (timed by the administration time), and the supernatant culture medium was discarded; Incubate at 37°C in a CO2 incubator for 48-72 hours until the cell fusion rate reaches more than 90% under a microscope; then discard the culture supernatant and wash each well twice with 1ml sterile PBS buffer; discard the PBS buffer and add 200μl of trypsin solution to each well for digestion for 3min; then add 1ml of PBS buffer to each well to blow the digested cells, and pipette the cell suspension into a 1.5ml EP tube, centrifuge at 300g for 5min, and discard the supernatant; add 150μl of melanin extract to each tube and place in a 90°C water bath for 1h to completely dissolve the melanin. Pipette 100μl of solution from each EP tube into a 96-well plate, use the melanin extract as the zero control, detect the absorbance value at 405nm with an enzyme reader, and use the size of OD405 to represent the content of melanin to calculate the melanin synthesis inhibition rate. The calculation formula is as follows: ; Table 2 Test sample addition table ; The results are as follows Figure 6As shown in the results, T-COL17R3 can reduce the synthesis of melanin in B16-F10 cells, and the inhibitory effect increases with the increase of concentration. When the concentration increases from 10μg / ml to 20μg / ml, the inhibitory effect on melanin is significantly improved. In addition, at the same concentration, the inhibitory effect of T-COL17R3 on melanin is better than that of T-COL17-NC16.

[0051] 2.2.2. Transdermal performance test of recombinant humanized type XVII collagen In order to test the transdermal absorption ability of recombinant humanized type XVII collagen, this paper used the static diffusion cell method to test the transdermal absorption ability of recombinant humanized type XVII collagen, and compared it with a similar product on the market as a positive control group. The specific steps are as follows: Take the pigskin of a 1-month-old Bama pig and fix it, with the stratum corneum facing up and the dermis facing down, and install the diffusion cell (schematic diagram of the diffusion cell, as shown in the figure). Figure 7 (as shown), the porcine skin sample was kept in contact with the liquid surface of the receiving cell; 6.5 ml of PBS was added to the receiving chamber, and 1 ml of each solution shown in Table 3 was added to the donor chamber. The diffusion instrument was started, and the duration was set to two groups of 24 h and 48 h, with three parallel samples in each group. The collagen content in the receiving cell at each time point was detected by the BCA assay.

[0052] Table 3 Solution information of each group ; from Figure 8 、 Figure 9 It can be seen that in the experimental group, the content of recombinant humanized type XVII collagen T-COL17R3 in the receiving pool was significantly higher than that of T-COL17-NC16 and similar commercial products after 24 hours and 48 hours. Therefore, the transdermal ability of the recombinant humanized type XVII collagen T-COL17R3 provided by the present invention is significantly improved compared with T-COL17-NC16 and similar commercial products.

[0053] 2.3 Anti-wrinkle and firming efficacy test 2.3.1. Anti-non-enzymatic glycosylation ability test The NBT method was used to detect the content of ketoamine, an early glycation product, and the Girard-T method was used to detect the content of dicarbonyl compounds, a late glycation product. The specific steps are as follows: (1) Solution preparation MGO stock solution: Prepare a 40 mM solution in ultrapure water and store at 5°C ± 3°C.

[0054] BSA stock solution: Prepare a 20 mg / ml solution in ultrapure water and store at 5°C ± 3°C.

[0055] (MGO + BSA) working solution: Accurately measure MGO (40mM) and BSA (20mg / ml) in a 1:1 ratio to prepare 20mM MGO + 10mg / ml BSA. Prepare immediately before use.

[0056] NBT working solution: Weigh NBT and prepare a 0.3 mM NBT solution using a pH 10.35 buffer solution.

[0057] Girard-T working solution: Weigh Girard-T and prepare a 500 mM Girard-T solution with ultrapure water.

[0058] Sodium formate working solution: Weigh sodium formate solid and prepare a 500 mM sodium formate solution with acetate buffer at pH 3.7.

[0059] Positive control AG (aminoguanidine) stock solution: Weigh an appropriate amount of AG, add ultrapure water to prepare 5 mg / ml, and store at 5℃±3℃.

[0060] Positive control AG working solution: Accurately measure an appropriate amount of AG stock solution (5 mg / ml) and prepare a series of concentration gradient solutions at 1000 ug / ml, 500 ug / ml, 250 ug / ml, 125 ug / ml, and 62.5 ug / ml to verify the system.

[0061] (2) Prepare the reaction system according to Table 4-5, and test after reacting at 37℃ for 5 days.

[0062] Table 4 Experimental sample addition table (ml) ; Table 5 Experimental sample addition table (ml) ; The results are as follows Figure 10 , Figure 11 and Figure 12 As shown, T-COL17R3 and T-COL17-NC16 have comparable abilities to resist ketamines, an early glycation product, and dicarbonyl compounds, a late glycation product, and their abilities far exceed those of similar commercial products at the same concentration.

[0063] 2.3.2 Anti-HACAT cell glycosylation ability test To further verify the anti-non-enzymatic glycation activity of the recombinant protein, the present invention used methylglyoxal (MGO) to induce non-enzymatic glycation in keratinocytes and further constructed a non-enzymatic glycation model at the cellular level. The specific steps are as follows: (1) After confirming that the cell confluence reaches 80%~90% under a microscope, remove the culture supernatant; (2) Treat the cells according to the cell passage method to obtain a single cell suspension, aspirate 100 μL of the cell suspension for cell counting, and adjust the cell density to 1×10 5 / mL; (3) Inoculate the adjusted cell suspension into a 96-well plate at 100 μL / well, and add sterile PBS buffer to the edge wells to prevent evaporation; (4) Place the inoculated cell culture plate in a constant temperature incubator and culture for about 24 hours; (5) Discard the culture supernatant, add MGO inducer solution prepared in DMEM, and continue incubating in a 5% CO2, 37℃ incubator for 24 hours; (6) Remove the inducer solution and add serum-free culture medium containing different concentrations of type XVII collagen and positive control AG, and incubate at 37°C for 48 hours; (7) Observe the changes in cell morphology under a microscope, wash the cells twice with sterile PBS, add 100uL of culture medium and 10uL of CCK-8 solution to each well, incubate at 37°C for 60 minutes, and detect the absorbance at 450nm using an enzyme-labeled instrument.

[0064] The results are as follows Figure 13 As shown, in the cell model, the cell proliferation rate in the methylglyoxal-induced group decreased to 55.24%, significantly lower than that in the group without MGO, indicating that MGO successfully induced cell damage and the successful establishment of the cell model. Based on this model, the anti-nonenzymatic glycation ability of the two recombinant collagens at the cellular level was explored. The experimental results showed that both recombinant proteins exhibited significant cytoprotective effects, effectively inhibiting cellular nonenzymatic glycation reactions, thereby delaying skin aging and improving skin wrinkles and sagging. At a concentration of 10 μg / mL, the anti-glycation effect of T-COL17R3 was superior to that of similar commercial products, but inferior to that of T-COL17-NC16.

[0065] 2.3.3 Antioxidant activity test Take 50 μl of recombinant humanized type XVII collagen of different concentrations, positive control Trolox (a water-soluble vitamin E derivative), and similar commercial products, add 150 μl of ABTS working solution (0.74 mmol / L ABTS, 0.26 mmol / L K2S2O8), incubate at room temperature for 6 minutes, measure the absorbance at 734 nm, calculate the scavenging effect of the samples on ATBS free radicals, and draw the scavenging rate curve.

[0066] The results are as follows Figure 14 and Figure 15As shown, T-COL17R3 and T-COL17-NC16 have comparable free radical scavenging abilities, which are concentration-dependent and far higher than similar commercially available products. This suggests that T-COL17R3, further proposed in the present invention, has potential anti-wrinkle and firming effects.

[0067] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A multi-effect repair collagen, characterized in that: The multi-functional repair collagen T-COL17R3 includes transdermal peptide TD-1, a partial functional fragment of type XVII collagen and a His tag. The partial functional fragment of type XVII collagen is selected from the transmembrane region, the extracellular sixteenth non-collagen region and the extracellular fifteenth collagen region of type XVII collagen, and the amino acid sequence is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that Encodes the multifunctional repair collagen T-COL17R3 according to claim 1, wherein the nucleotide sequence of the multifunctional repair collagen T-COL17R3 is shown in SEQ ID NO.

3.

3. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the polynucleotide sequence according to claim 2 and can correspondingly translate and express the multi-functional repair collagen T-COL17R3 according to claim 1.

4. A host cell, characterized in that The host cell contains the recombinant plasmid according to claim 4 or expresses the multi-functional repair collagen T-COL17R3 according to claim 1.

5. A host cell according to claim 4, characterized in that The cells include Pichia pastoris.

6. A method for preparing the multi-effect repair collagen according to claim 1, characterized in that: The following steps are involved: (1) inoculating the host cell according to any one of claims 4 to 5 into a fermentation medium for fermentation culture, and after the fermentation culture is completed, centrifuging and collecting the supernatant to obtain a fermentation broth; (2) The fermentation liquid obtained in step (1) is separated and purified, and the protein is detected to obtain the multi-functional repair collagen.

7. Use of the multi-functional repair collagen according to claim 1 in the preparation of a pharmaceutical composition or a skin care product.

8. The use according to claim 7, characterized in that The pharmaceutical composition or skin care product has at least one of the effects of repairing, anti-wrinkle and firming, or whitening and brightening.

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