Recombinant human collagen and application thereof

Abstract

The invention relates to recombinant human collagen and application thereof, in particular to application of combination of collagen and ascorbic acid polypeptide derivatives in preparation of anti-photoaging, anti-aging and anti-oxidation drugs or cosmetics or skin care products.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a gene recombinant human collagen and a use thereof. Background Art

[0002] Collagen is a glycoprotein. It is divided into more than 28 types of collagen according to different tissue locations, physiological functions, and molecular structures. The most studied and thorough types are type I, type II, and type III collagen. There are many types of collagen, and the common types are type I, type II, type III, type V, and type XI. The collagen in human skin is type I collagen and type III collagen. Type I collagen is mainly found in adult skin, tendons, and bone tissues. Type III collagen is mainly found in infant skin or vascular endothelium and intestines. Type I collagen and type III collagen are closely related to the skin damage repair process and repair quality. Among various collagens, type 21 collagen is a non-fibrous collagen that is relatively rare in the adult human body, accounting for less than 1% of the total collagen. Compared with other more well-known types of collagen, research on type 21 collagen is relatively less.

[0003] L-Ascorbic acid is a natural water-soluble antioxidant whitening agent, also known as vitamin C, and is widely used as an anti-aging ingredient in cosmetics. However, its sensitivity to light and oxygen and unstable structure limit its application in the cosmetics industry. Patent application WO2022228475A1 records an ascorbic acid polypeptide derivative, a new structure of ascorbic acid polypeptide derivative, which solves the stability problem of the traditional VC structure, and its content is fully incorporated into this application.

[0004] The inventors of the present application unexpectedly discovered that the combined use of L-ascorbic acid derivatives and collagen can significantly improve the ability of cells to resist oxidation and photoaging, and has a significantly higher effect than the use of L-ascorbic acid derivatives or collagen alone, showing a clear synergistic effect. In addition, even if collagen or L-ascorbic acid derivatives are used alone, they also have certain above-mentioned effects. Summary of the invention

[0005] In order to solve the defects of the prior art, the technical problem solved by the present invention is to provide a genetically recombinant human collagen protein, the amino acid sequence of which is shown in SEQ ID NO.7:

[0006] GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARG

[0007] LPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKK

[0008] GAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTR

[0009] GEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG.

[0010] In some embodiments, the collagen is recombinant humanized collagen type 21;

[0011] In another aspect, the present invention relates to a vector containing the protein encoding gene;

[0012] In another aspect, the present invention relates to a host cell of the protein encoding gene or the vector;

[0013] On the other hand, the present invention relates to the use of the recombinant human collagen, the vector, and the host cell in preparing cosmetics, health products, or medicines;

[0014] In another aspect, the present invention relates to a method for producing the recombinant human collagen, which is characterized by comprising the following steps: (1) constructing genetically engineered Pichia pastoris; (2) fermenting and culturing the genetically engineered Pichia pastoris; (3) inducing and expressing the recombinant human collagen; (4) purifying the recombinant human collagen;

[0015] In another aspect, the present invention provides an ascorbic acid polypeptide derivative, a compound represented by the following general formula (1) or a salt thereof:

[0016]

[0017] Wherein, m is 0 and n is 1, or, m is 1 and n is 0;

[0018] X1 and X2 are independently C1-C6 alkyl, C1-C6 alkoxy or halogen;

[0019] Q1 and Q2 are independently C1-C6 alkyl chains;

[0020] a, b, c are independently selected from integers of 1-10;

[0021] e is 0 and d is an integer of 1-10, or e is 1 and d is 0.

[0022] Furthermore, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (3):

[0023]

[0024] Furthermore, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (4):

[0025]

[0026] Furthermore, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (6):

[0027]

[0028] In a second aspect, the present invention provides a method for preparing the above-mentioned ascorbic acid polypeptide derivative, comprising: first activating the carboxyl group of a raw material A having an amino protecting group, then reacting with a raw material B, and obtaining the ascorbic acid polypeptide derivative after removing the protecting group;

[0029] Wherein, the raw material A is a compound represented by the following general formula (7):

[0030]

[0031] a and b are independently selected from integers of 1 to 10;

[0032] The raw material B is a compound represented by the following general formula (8):

[0033]

[0034] k is an integer selected from 1 to 10; Q is a C1 to C6 alkyl chain;

[0035] Y is a C1-C6 alkyl group, a C1-C6 alkoxy group or a halogen group.

[0036] Further, the raw material A is carnosine having an amino protecting group; the raw material B is 3-O-ethyl ascorbic acid;

[0037] In some embodiments, the ascorbic acid polypeptide derivative is a compound represented by the following general formula (3), formula (4), and formula (6):

[0038]

[0039] On the other hand, the present invention relates to the use of collagen in the preparation of a medicine or cosmetic or skin care product for use in combination with a second composition having repair and restoration effects, anti-photoaging, anti-aging and anti-oxidation, wherein the second composition is an ascorbic acid polypeptide derivative.

[0040] In some embodiments, the collagen is type 21 collagen.

[0041] In some embodiments, the amino acid sequence of the collagen is as shown in SEQ ID NO.7.

[0042] In some embodiments, the present invention relates to a composition comprising 0.002% of the aforementioned collagen and 0.0002% of the aforementioned ascorbic acid polypeptide derivative.

[0043] In some embodiments, the present invention relates to a composition consisting of 0.002% of the aforementioned collagen and 0.0002% of the aforementioned ascorbic acid polypeptide derivative.

[0044] In some embodiments, the present invention relates to a composition consisting essentially of 0.002% of the aforementioned collagen and 0.0002% of the aforementioned ascorbic acid polypeptide derivative. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Experimental results of anti-aging.

[0046] Figure 2 : Experimental results of barrier antioxidant repair. DETAILED DESCRIPTION

[0047] definition:

[0048] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0049] Example 1: Synthesis of ascorbic acid polypeptide derivatives

[0050] 1. First, Boc-L-Carnosine (13 g, 39.9 mmol) was dissolved in DMF (400 mL);

[0051] 2. Next, add DCC (7.98 g, 38.8 mmol) and HOSu (4.45 g, 38.8 mmol);

[0052] 3. Then, the solution was stirred at room temperature for about 15 hours, and the solid by-product (N,N-dicyclohexylurea) was removed after filtration.

[0053] 4. Then, add 3-O-ethyl ascorbic acid ether (8.2 g, 40.2 mmol) and DIPEA (16 mL, 92 mmol) to the filtrate;

[0054] 5. Subsequently, the mixture was stirred at room temperature for 3 h, then concentrated and dried in vacuo;

[0055] 6. The solid was recrystallized from methanol and isopropyl ether to obtain Boc-EAC-L-Carnosine (16.4 g, 32 mmol) as a light yellow solid;

[0056] 7. Dissolve Boc-EAC-L-Carnosine in 1,4-dioxane (100 mL), add 4M hydrochloric acid and 1,4-dioxane (99 mL, 396 mmol);

[0057] 8. The mixture was stirred at room temperature for 4 h, then concentrated, and the solid was triturated with acetone and isopropanol to purify EAC-L-Carnosine (3-O-ethyl ascorbic acid-carnosine) (5.2 g, 98%);

[0058] 9. After freeze-drying, EAC-L-Carnosine is a white powder (yield 4.9%).

[0059] The synthesis process (the relevant content of CN115260170A is quoted in full) is as follows:

[0060]

[0061] Example 2: Synthesis process of collagen

[0062] 1. Construction of genetically engineered Pichia pastoris

[0063] pPic9k was used as a vector to construct a recombinant plasmid, and the gene sequence of the type 21 collagen fragment GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG was inserted to obtain the type 21 collagen vector.

[0064] Activate yeast GS115 and streak culture on YPD plate, select single clone of Pichia GS115 activated on YPD plate and culture in YPD liquid, shake and culture at constant temperature; pipette 100uL bacterial solution and inoculate it in 100mL YPD liquid, 30℃, 220rpm, 12-13h, culture until OD 600 =1.3-1.5; observe under a microscope for bacterial contamination, divide the bacteria into sterile 50ml centrifuge tubes and place on ice for 10-15 minutes.

[0065] Centrifuge twice, discard the supernatant, and resuspend the bacteria in sterile water; centrifuge twice more, discard the supernatant, resuspend the bacteria in sorbitol and then package them to obtain competent cells; use electroporation to transfer the vector into competent cells, and then culture on YPD plates until clones are produced.

[0066] 2. Fermentation of genetically engineered Pichia pastoris

[0067] A single colony was picked from the YPD plate and placed in a conical flask containing 50 mL of BMGY. The culture was carried out at 30°C and 220 rpm for 24 h. The OD 600 The culture was completed; centrifuged for 10 min, the cells were collected, washed once with BMMY, centrifuged again, and the cell concentration was diluted to OD 600 =1.0.

[0068] 3. Induction and expression of recombinant human collagen

[0069] Transfer the diluted bacterial solution to a 500mL conical flask and induce yeast to express protein at 29℃ and 220rpm. Then add methanol every 24h to make the final concentration of methanol in the culture medium 1% and continue inducing culture for 84h. Check under a microscope to see if there is any bacterial contamination and measure the pH of the fermentation liquid, which should be less than 6. Transfer the fermentation liquid to a 500mL round-bottom centrifuge tube and centrifuge at 15000g and 4℃ for 20min to remove the supernatant. Remove 1mL of supernatant and add corresponding loading buffer. Mix well and incubate at 95℃ for 10min and store at -20℃.

[0070] 4. Purification of recombinant human collagen:

[0071] Transfer the obtained fermentation broth supernatant to a conical flask, add NaCl to make the final concentration of NaCl 150mM, adjust the pH of the fermentation broth supernatant to 8.0 with NaOH or HCl; centrifuge at 15000g for 30min to ensure that the supernatant is clear and transparent, balance the column with 10 nickel column bed volumes of binding buffer; load the sample at a speed of 1mL / min, and collect the column liquid while loading the sample. Elute the unbound protein and impurity protein with 5 nickel column bed volumes of binding buffer; elute the target protein with elution buffer (500mM imidazole).

[0072] Preparation of protein detection samples: Add the collected purified proteins, supernatant before column penetration, and liquid after column penetration into the corresponding loading buffer, mix well, incubate at 95℃ for 10min, and store at -20℃.

[0073] Through this series of steps, recombinant humanized collagen type 21 was successfully prepared and purified, and the Pichia pastoris expression system was used to obtain a high-purity, high-activity target protein.

[0074] Among them, after sequencing confirmation, the amino acid sequence of the collagen is shown as SEQ ID NO:7.

[0075] GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGL PGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG

[0076] Example 3: Experiment on photoaging and anti-aging

[0077] 4.1 Sample

[0078] Type 21 collagen solution: a solution containing 10000 ppm of the target recombinant type 21 collagen molecule (amino acid sequence as shown in SEQ ID NO: 7)

[0079] Carnosine C solution: a solution containing 1000ppm EAC-L-Carnosine target molecule

[0080] 4.2 Experimental Group

[0081] GroupA 0.2% type 21 collagen solution (ie: 0.002% type 21 collagen)

[0082] Group B 0.2% Carnosine C solution (ie: 0.0002% EAC-L-Carnosine target molecule)

[0083] Group C 0.1% type 21 collagen solution + 0.1% myopeptide C solution

[0084] Group D 0.16% type 21 collagen solution + 0.04% myopeptide C solution

[0085] The experimental cells were HFF-1 human skin fibroblast-like cells, which were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0086] Culture conditions:

[0087] The cells were cultured in a 5% CO2, 37°C constant temperature incubator.

[0088] The culture medium was 89% DMEM+10% FBS+1% double antibody.

[0089] Reagent preparation:

[0090] The test substances and control substances were prepared into stock solutions with DMSO or ddH2O and then diluted to appropriate concentrations with serum-free culture medium or PBS.

[0091] Steps:

[0092] qPCR detection:

[0093] Cells in the exponential growth phase were made into single-cell suspensions and seeded in 6-well plates at a density of 1×10^5 cells / mL.

[0094] The cells were cultured in a medium containing 10% fetal bovine serum for 24 hours, and then the supernatant was discarded. Serum-free medium or serum-free medium containing different concentrations of drugs was added and incubated for 48 hours.

[0095] After RNA was extracted, the expression of Col-I and Elastin mRNA was detected by qPCR.

[0096] The primer information is as follows:

[0097]

[0098] Experimental results (see Figure 1and Table 1) show that Group C is significantly better than Group A and Group B. In other words, the expression of collagen and elastin produced by the combined use of type 21 collagen solution and myoglobin C solution is significantly higher than that of type 21 collagen solution or myoglobin C solution alone. The widely used Burgi formula method is used to calculate synergy: q = E〔A / 2+B / 2〕 / EA (or EB), where E〔A / 2+B / 2〕 refers to the effect of half the amount of A and B, and EA and EB are the effects of A and B alone, respectively. The q calculated by the above formula is greater than 1, which proves that the two have a synergistic effect and have achieved an unexpected synergistic effect.

[0099] Table 1: Experimental results

[0100]

[0101] Example 4: Experiment on barrier antioxidant repair

[0102] 5.1 Sample

[0103] Type 21 collagen solution: a solution containing 10000 ppm of recombinant type 21 collagen target molecule (SEQ ID NO: 7)

[0104] Carnosine C solution: a solution containing 1000ppm EAC-L-Carnosine target molecule

[0105] 5.2 Experimental Group

[0106] GroupA 0.2% type 21 collagen solution (ie: 0.002% type 21 collagen)

[0107] Group B 0.2% Carnosine C solution (ie: 0.0002% EAC-L-Carnosine target molecule)

[0108] Group C 0.1% type 21 collagen solution + 0.1% myopeptide C solution

[0109] Group D 0.16% type 21 collagen solution + 0.04% myopeptide C solution

[0110] Cell seeding:

[0111] The cells were diluted to a seeding density of 3×10^5 cells / mL. 100 μL of the cell dilution was used to seed a 96-well plate and incubated in a cell culture incubator (37° C., 5% CO 2 , 95% RH) for 24±2 hours.

[0112] Experimental groups:

[0113] A zero adjustment group, a normal group, a H2O2 modeling group, and a sample group were set up, with 3 replicate wells in each group.

[0114] For ROS content detection, a normal group, H2O2 modeling group, positive control group and sample group were set up, and 3 replicate wells were set up in each group.

[0115] Liquid preparation:

[0116] Sample solution: Dissolve the sample in cell culture medium (DMSO can be added to aid dissolution for samples that are difficult to dissolve, but the final concentration of DMSO should not exceed 0.5%), and further dilute with cell culture medium.

[0117] H2O2 working solution: Prepare the H2O2 stock solution into the required working solution using serum-free culture medium.

[0118] CCK-8 working solution: Add 1 mL of CCK-8 stock solution to 9 mL of serum-free medium and mix well to prepare 10 mL of CCK-8 working solution.

[0119] H2O2 Induction:

[0120] When the cell confluence in the 96-well plate reached 50% to 70%, the cell culture medium of each group was aspirated, washed once with PBS and discarded, and H2O2 working solution was added.

[0121] Add sample:

[0122] After induction, the cells were washed twice with PBS. 100 μL of culture medium was added to each well of the normal group; 100 μL of culture medium containing samples of corresponding concentrations was added to each well of the sample group; and no cell inoculation was performed in the zero adjustment group, and only 100 μL of cell culture medium was added.

[0123] After the sample addition was completed, the 96-well plate was placed in a cell culture incubator (37° C., 5% CO 2 , 95% RH) for 24±2 hours.

[0124] Cell viability assay:

[0125] After culturing for 24±2 hours, first observe the cell status under a microscope, then discard the supernatant, add 100 μL of CCK-8 working solution to each well, and incubate at 37°C in the dark for 2±0.5 hours.

[0126] After the incubation, the optical density (OD value) was read at a wavelength of 450 nm. Cell activity (%) = (OD of sample group - OD of zero-adjustment group) / (OD of blank group - OD of zero-adjustment group) × 100%.

[0127] The experimental results show that (see Figure 2and Table 2): Similar to the conclusion of Example 4, in the experiment of barrier antioxidant repair, a synergistic effect was also achieved. Specifically, in the modeling environment, the cell activity of the combined drug administration group (e.g., Group C) was significantly better than that of the single drug administration group, and this difference was significant. The widely used Burgi formula method was used to calculate the synergy: q = E〔A / 2+B / 2〕 / EA (or EB), where E〔A / 2+B / 2〕 refers to the effect of half the amount of A and B drugs, and EA and EB are the effects of A and B alone, respectively. The q calculated by the above formula is greater than 1, which proves that the two have a synergistic effect and an unexpected synergistic effect has been achieved.

[0128] Table 2: Experimental results

[0129]

[0130] The above description is only a preferred embodiment, which is only used as an example and does not limit the combination of features necessary to implement the present invention. The titles provided are not intended to limit the various embodiments of the present invention. Terms such as "comprising", "including" and "including" are not intended to be limiting. In addition, unless otherwise specified, plural forms are included when there is no numeral modification, and "or" means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

Claims

1. A recombinant human collagen, characterized in that The amino acid sequence is shown in SEQ ID NO.

7.

2. A vector containing the protein encoding gene according to claim 1.

3. A host cell containing the protein encoding gene according to claim 1 or the vector according to claim 2.

4. Use of the recombinant human collagen according to claim 1, the vector according to claim 2, and the host cell according to claim 3 in the preparation of cosmetics, health products or medicines with anti-photoaging, anti-aging and antioxidant effects.

5. Use of the collagen according to claim 1 in the preparation of a drug or cosmetic or skin care product for use in combination with a second composition for anti-photoaging, anti-aging and anti-oxidation, wherein the second composition is an ascorbic acid polypeptide derivative.

6. The use according to claim 5, wherein the content of collagen is 0.002%, and the content of ascorbic acid polypeptide derivative is 0.0002%.

7. The use according to any one of claims 5 to 6, wherein the ascorbic acid polypeptide derivative is a compound represented by the following general formula (3), formula (4), or formula (6):

8. A composition comprising 0.002% collagen and 0.0002% ascorbic acid polypeptide derivative, wherein the amino acid sequence of the collagen is as shown in SEQ ID NO.7, and the ascorbic acid polypeptide derivative is a compound represented by the following general formula (3), formula (4), and formula (6):

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