A transdermal multi-type recombinant collagen, a recombinant collagen solution, and a preparation method and application thereof

Abstract

The present application relates to the technical field of bioengineering, and specifically discloses a transdermal multi-type recombinant collagen, a recombinant collagen solution, and a preparation method and application thereof. The recombinant collagen includes type I collagen, type II collagen, type III collagen, type IV collagen, and type XVII collagen, and the copies of each type of collagen are connected by sequences that can be recognized and cut by proteases; the N segment or C end of the recombinant collagen is connected with a transdermal short peptide through a linker sequence. The sequences of the above-mentioned types of collagen include the amino acid sequences shown in SEQ ID NO.1‑5, respectively. The transdermal multi-type recombinant collagen provided in the present application can simultaneously provide multiple types of collagen required by the skin, and provide supplementary nutrition for the skin; in addition, the synthesis of multiple collagens is achieved by a one-step method, which greatly reduces the preparation cost of collagen compared with the traditional composite method.

Description

Technical Field

[0001] The present application relates to the technical field of bioengineering, and more specifically, to a transdermal multi-type recombinant collagen, a recombinant collagen solution, and a preparation method and application thereof. Background Art

[0002] Collagen is a fibrous protein composed of three peptide chains entangled with each other. It is widely present in the connective tissue of animals and is the most widely distributed functional protein in mammals. So far, researchers have discovered 28 types of collagen, which can be divided into fibroblastic collagen and non-fibroblastic collagen according to their structure and function. Common fibroblastic collagens include type I, type II, type III, type IV, type V, type VI, and type XI; while non-fibroblastic collagen is type XVII collagen (COL17), including reticular collagen, anchoring collagen, and transmembrane collagen. Type XVII collagen plays an important role in the function of the basement membrane of epithelial cells, including regulating the adhesion, separation, and developmental differentiation of epithelial cells, as well as maintaining the homeostasis of related stem cells in the skin and hair growth. Therefore, type XVII collagen plays a great role in delaying skin aging and promoting hair follicles. Collagen type I, composed of tightly packed fibers, accounts for 90% of the collagen in the human body and provides structure for skin, bones, tendons, fibrocartilage, connective tissue and teeth. Collagen type III is secreted by fibroblasts and other mesenchymal cell types, making it a major player in various inflammatory-driven pathologies, such as lung injury, viral and non-viral liver disease. Collagen type IV is the main type of collagen found in the basement membrane. It is a network-forming collagen that is the basis of epithelial and endothelial cells and can serve as a barrier between tissue compartments. Studies have shown that a large number of free radicals produced by ultraviolet (UV) radiation have a damaging effect on macromolecules in the body and can cause skin cell damage and apoptosis. As the ozone layer gradually thins, the intensity of human exposure to ultraviolet radiation continues to increase, and the number of diseases caused by ultraviolet radiation is increasing. For example, the incidence of skin cancer and cataracts has increased significantly in recent years. Type VI collagen, as a structural protein, can stimulate interstitial cells to synthesize increased DNA and promote cell proliferation. Soluble type VI collagen can significantly promote cell migration and distribution. Type VI collagen may have growth factor-like properties. During inflammation or remodeling, it may be one of the first extracellular matrix (ECM) molecules to be digested and released by proteases. These released type VI collagen fragments can promote interstitial cell mitosis and rapidly repair wounds as autocrine or paracrine mediators. When targeting cell apoptosis caused by DNA damage, type VI collagen can protect cells from UV-induced cell apoptosis.

[0003] As a natural biopolymer material, collagen has good biocompatibility, biodegradability and absorbability, and can promote cell formation and epithelial cell formation. It can be widely used in medicine, beauty, cosmetics, health care products and other fields. Its role in skin damage repair, plastic surgery, and enhancing the body's immunity has also been recognized. At present, the sources of collagen mainly include animal extraction and genetic engineering. Animal-extracted collagen may have zoonotic diseases, so there may be certain risks in its use; recombinant collagen derived from genetic engineering is currently a widely recognized traditional collagen substitute product.

[0004] In order to more comprehensively supplement collagen for the skin, technicians usually add multiple types of collagen to the formula and apply it to the skin surface in the form of a collagen composition, thereby achieving the purpose of comprehensively supplementing the collagen components required by the skin. However, in the above case, different types of collagen need to be prepared separately, which makes the preparation process and production cost of collagen application relatively high. Summary of the invention

[0005] In order to provide low-cost and rich collagen, the present application provides a transdermal multi-type recombinant collagen, a recombinant collagen solution, and a preparation method and application thereof.

[0006] This application uses gene fusion to connect 5 different DNA fragments, transdermal short peptides that can promote transdermal penetration of molecules, and specific enzyme cleavage sites into a fusion protein composed of 5 different collagen helical domains. Through microbial fermentation expression, 5 different types of collagen fragments are cut out using proteases. The composite peptide does not need to be compounded, has a low preparation cost, and can be effectively absorbed by the skin.

[0007] In the first aspect, the present application provides a transdermal multi-type recombinant collagen protein, using the following technical solution:

[0008] A transdermal multi-type recombinant collagen, wherein the recombinant collagen comprises type I collagen, type III collagen, type IV collagen, type VI collagen and type XVII collagen;

[0009] The type I collagen comprises the amino acid sequence shown in SEQ ID NO.1, and the copy number is m, m≥1;

[0010] The type II collagen includes the amino acid sequence shown in SEQ ID NO.2, and the copy number is n, n≥1;

[0011] The type III collagen comprises the amino acid sequence shown in SEQ ID NO.3, and the copy number is o, o≥1;

[0012] The type IV collagen includes the amino acid sequence shown in SEQ ID NO.4, and the copy number is p, p≥1;

[0013] The type XVII collagen includes the amino acid sequence shown in SEQ ID NO.5, and the copy number is q, q≥1;

[0014] The type I collagen, the type II collagen, the type III collagen, the type IV collagen, the type XVII collagen, and the number of copies of each type of collagen are all connected by sequences that can be recognized and cleaved by proteases;

[0015] The N segment or C terminus of the recombinant collagen is connected with a transdermal short peptide via a linker sequence; the transdermal short peptide comprises an amino acid sequence as shown in SEQ ID NO.6.

[0016] Optionally, the copy number ratio of the type I collagen, the type II collagen, the type III collagen, the type IV collagen and the type XVII collagen is:

[0017] m:n:o:p:q=(6-14):(2-6):(2-10):(1-6):(1-6).

[0018] Optionally, the copy number ratio of the type I collagen, the type II collagen, the type III collagen, the type IV collagen and the type XVII collagen is:

[0019] m:n:o:p:q=(8-10):(3-4):(4-8):(2-3):(2-3).

[0020] In a specific embodiment, in the above copy number ratio, the value corresponding to type I collagen can be 6, 8, 10, or 14.

[0021] In some specific embodiments, in the above copy number ratio, the numerical range corresponding to type I collagen can be 6-8, 6-10, 6-14, 8-10, 8-14, 10-14.

[0022] In a specific embodiment, in the above copy number ratio, the value corresponding to type II collagen can be 2, 3, 4, or 6.

[0023] In some specific embodiments, in the above copy number ratio, the numerical range corresponding to type II collagen can be 2-3, 2-4, 2-6, 3-4, 3-6, 4-6.

[0024] In a specific embodiment, in the above copy number ratio, the value corresponding to type III collagen can be 2, 4, 6, 8, or 10.

[0025] In some specific embodiments, in the above copy number ratio, the numerical range corresponding to type III collagen can be 2-4, 2-6, 2-8, 2-10, 4-6, 4-8, 4-10, 6-8, 6-10, 8-10.

[0026] In a specific embodiment, in the above copy number ratio, the value corresponding to type IV collagen can be 1, 2, 3, 4, or 6.

[0027] In some specific embodiments, in the above copy number ratio, the numerical range corresponding to type IV collagen can be 1-2, 1-3, 1-4, 1-6, 2-3, 2-4, 2-6, 3-4, 3-6, 4-6.

[0028] In a specific embodiment, in the above copy number ratio, the value corresponding to type XVII collagen can be 1, 2, 3, 4, or 6.

[0029] In some specific embodiments, in the above copy number ratio, the numerical range corresponding to type XVII collagen can be 1-2, 1-3, 1-4, 1-6, 2-3, 2-4, 2-6, 3-4, 3-6, 4-6.

[0030] Optionally, the recombinant collagen comprises an amino acid sequence as shown in SEQ ID NO.7-SEQ ID NO.10.

[0031] Optionally, the protease is selected from TEV protease, EK protease, collagenase or Thrombin.

[0032] Optionally, the protease is TEV protease.

[0033] In a second aspect, the present application provides a gene sequence encoding the above-mentioned transdermal multi-type recombinant collagen.

[0034] In a third aspect, the present application provides a method for preparing a recombinant collagen solution, using the following technical solution:

[0035] A method for preparing a recombinant collagen solution, the preparation method specifically comprising the following steps:

[0036] The gene sequence corresponding to the above-mentioned transdermal polymorphic recombinant collagen is designed, a recombinant vector containing the gene sequence is constructed, and the gene sequence is introduced into competent cells to screen for high-expression strains of the polymorphic recombinant collagen; the polymorphic recombinant collagen is prepared using the high-expression strain, and the recombinant collagen solution is obtained by purification.

[0037] In a fourth aspect, the present application provides a recombinant collagen solution prepared using the above-mentioned preparation method.

[0038] In a fifth aspect, the present application provides an application of the above-mentioned recombinant collagen solution in tissue repair, scar healing, cell repair, anti-aging and skin moisturizing.

[0039] In summary, this application has the following beneficial effects:

[0040] 1. The first purpose of the present application is to provide a transdermal molecular design of multiple recombinant collagens to solve the problems of low yield, low stability, low homology, low purity, high impurity content, and high risk of allergens obtained by existing separate culture systems of various types of collagens. In addition, since type VI and type XVII collagens contain a high proportion of rare codons when cultured alone, they are prone to low or even no expression levels. When they are connected with other types of collagens through genetic engineering to form a long chain for collaborative culture, shared codons can increase their protein expression and increase yield.

[0041] 2. The second objective of the present application is to provide a method for preparing transdermal multi-type recombinant collagen.

[0042] 3. The third objective of the present application is to provide a transdermal application of multi-type recombinant collagen.

[0043] 4. The transdermal multi-type recombinant collagen obtained in the present application can promote the collagen to pass through the skin barrier by means of the transport effect of the transdermal short peptides contained therein, thereby improving the skin utilization of collagen and replenishing collagen for the skin.

[0044] 5. The transdermal multi-type recombinant collagen provided in the present application has better support and cohesion, stronger biological activity and higher stability. The transdermal multi-type recombinant collagen can synergistically enhance the collagen reloading capacity, and can promote the secretion of new collagen by the nutrients of the extracellular matrix in the skin, thereby promoting the skin's regeneration and repair ability to restore the skin to a healthy state. DETAILED DESCRIPTION

[0045] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0046] The present application provides a transdermal multi-type recombinant collagen protein and a gene sequence for encoding the transdermal multi-type recombinant collagen protein.

[0047] The above-mentioned transdermal multi-type recombinant collagen includes type I collagen, type II collagen, type III collagen, type IV collagen and type XVII collagen. Among them, type I collagen includes the amino acid sequence shown in SEQ ID NO.1. Type II collagen includes the amino acid sequence shown in SEQ ID NO.2. Type III collagen includes the amino acid sequence shown in SEQ ID NO.3. Type IV collagen includes the amino acid sequence shown in SEQ ID NO.4. Type XVII collagen includes the amino acid sequence shown in SEQ ID NO.5. The copy number of each type of collagen can be arbitrary.

[0048] Type I collagen, type II collagen, type III collagen, type IV collagen, type XVII collagen and the number of copies of each type of collagen are connected by sequences that can be recognized and cut by proteases. The N segment or C end of the recombinant collagen is connected to a transdermal short peptide through a linker sequence; the transdermal short peptide includes an amino acid sequence as shown in SEQ ID NO.6.

[0049] Furthermore, the copy number of type I collagen is m, m≥1. The copy number of type II collagen is n, n≥1. The copy number of type III collagen is o, o≥1. The copy number of type IV collagen is p, p≥1. The copy number of type XVII collagen is q, q≥1.

[0050] Furthermore, the copy number ratios of type I collagen, type II collagen, type III collagen, type IV collagen and type XVII collagen are: m:n:o:p:q=(6-14):(2-6):(2-10):(1-6):(1-6).

[0051] The amino acid sequence of the above-mentioned recombinant collagen includes the amino acid sequences shown in SEQ ID NO.7 to SEQ ID NO.10.

[0052] The protease is selected from TEV protease, EK protease, collagenase or Thrombin.

[0053] In addition, the present application also discloses the gene sequence encoding the above-mentioned transdermal recombinant multi-type collagen.

[0054] The present application also provides a recombinant collagen solution and a preparation method thereof, the preparation method specifically comprising the following steps:

[0055] The gene sequence corresponding to the above-mentioned transdermal polymorphic recombinant collagen is designed, a recombinant vector containing the gene sequence is constructed, and the gene sequence is introduced into competent cells to screen for high-expression strains of the polymorphic recombinant collagen; the polymorphic recombinant collagen is prepared using the high-expression strain, and the recombinant collagen solution is obtained by purification.

[0056] The present application also provides the use of the above-mentioned recombinant collagen solution in tissue repair, scar healing, cell repair, anti-aging and skin moisturizing.

[0057] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application are described clearly and completely below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0058] The present application is further described in detail below in combination with embodiments, comparative examples and performance test results.

[0059] Example

[0060] Example 1

[0061] This embodiment provides a recombinant collagen.

[0062] The recombinant collagen includes the following parts:

[0063] (1) Type I collagen sequence, including the amino acid sequence shown in SEQ ID NO.1.

[0064] (2) Type II collagen sequence, including the amino acid sequence shown in SEQ ID NO.2.

[0065] (3) Type III collagen sequence, including the amino acid sequence shown in SEQ ID NO.3.

[0066] (4) Type IV collagen sequence, including the amino acid sequence shown in SEQ ID NO.4.

[0067] (5) Type XVII collagen sequence, including the amino acid sequence shown in SEQ ID NO.5.

[0068] (6) A transdermal short peptide comprising the amino acid sequence shown in SEQ ID NO.6.

[0069] Each type of collagen and the number of copies of each type of collagen are connected by a sequence that can be recognized and cleaved by TEV protease, the sequence is "EDLYFQ". The transdermal short peptide is connected to the C-terminus of the recombinant collagen in this example through a linker sequence, the linker sequence is "GGGGS".

[0070] In this embodiment, the copy numbers of various types of collagen are shown in Table 1.

[0071] In addition, Table 1 also lists the amino acid sequences of the recombinant collagen corresponding to some embodiments.

[0072] Table 1 The copy number of each type of collagen in each embodiment

[0073]

[0074]

[0075] This embodiment also provides the preparation process of the above-mentioned recombinant collagen high expression strain and recombinant plasmid.

[0076] The preparation process of the above-mentioned high expression strain of recombinant collagen and recombinant plasmid is as follows:

[0077] (1) Sequence construction

[0078] According to the amino acid sequence of the above recombinant collagen, the corresponding nucleotide sequence is designed and synthesized by gene synthesis to obtain the corresponding fusion gene fragment.

[0079] (2) Construction of expression vector

[0080] The fusion gene fragment was then inserted into the yeast vector pPic9k (purchased from Shanghai Biotechnology) to obtain a recombinant plasmid.

[0081] (3) Screening of high expression strains

[0082] Then, the recombinant plasmid obtained by linearization with sglII was purified by a DNA recovery kit and transferred into GS115 Pichia pastoris competent cells (purchased from Shanghai Biotechnology) by electroporation. Positive clones were screened by MD culture plates, and then high-expression strains of the recombinant fusion protein were screened by different concentrations of G418 (aminosugar antibiotics), thereby obtaining the above-mentioned high-expression strains of the recombinant collagen protein.

[0083] This embodiment also provides a preparation process of the above-mentioned recombinant collagen, and a purification process of the recombinant collagen solution composed of the above-mentioned recombinant collagen.

[0084] The preparation process of the above recombinant collagen is as follows:

[0085] The selected high-expression strains were inoculated into YPD medium and cultured overnight at 28°C and 220 rpm to prepare the revived seed solution. On the second day, 10 ml of the revived seed solution was transferred to 1 L of BMGY medium at a volume ratio of 1:100 and cultured at 28°C and 220 rpm until the OD 600 =2, and the cells were collected by centrifugation.

[0086] The cells were resuspended in BMMY medium, and the culture was continued at 28°C and 220 rpm with shaking, 1% (volume ratio) methanol was added every 24 hours, and samples were taken to detect the expression of recombinant collagen. The fermentation was terminated after 72 hours.

[0087] The purification process of the above recombinant collagen solution is as follows:

[0088] 1) After fermentation, add lactose (concentration of 200 g / L) and surfactant (TritonX100) to the fermentation broth and stir;

[0089] 2) separating the fermentation liquid after the treatment in step 1) by a high-speed centrifuge, removing the supernatant and collecting the precipitate, adding the precipitate to a hypotonic buffer solution with a pH of 6.0 to 7.5 containing EDTA, and stirring for 8 to 16 hours;

[0090] 3) separating the solid and liquid of the liquid treated in step 2) by a high-speed centrifuge, removing the supernatant and collecting the precipitate, and dissolving the precipitate in a buffer solution having a pH of 3.0-4.0;

[0091] 4) separating the solid and liquid of the liquid treated in step 3) by a high-speed centrifuge, removing the precipitate and collecting the supernatant;

[0092] 5) adding sodium chloride to the treated liquid in step 4) for salting out for 0.5 h, then separating the solid and liquid by a high-speed centrifuge, removing the supernatant and collecting the precipitate;

[0093] 6) dissolving the precipitate treated in step 5) in a buffer solution with a pH of 4.0-5.0, and adding copper sulfate and tyrosinase to carry out an enzymatic reaction;

[0094] 7) adding an acid solution to the treated solution in step 6) to adjust the pH of the solution to 3.0-4.0, and purifying the solution by cation exchange chromatography column equilibrated with mobile phase A, wherein mobile phase B1 is used to elute impurities, and mobile phase B2 is used to elute recombinant collagen;

[0095] Mobile phase A is an acid solution of pH 3.0-4.0, mobile phase B1 is an acid solution of pH 3.0-4.0 containing sodium chloride, and mobile phase B2 is an acid solution of pH 3.0-4.0 containing urea and sodium chloride; the acid solution is selected from one of hydrochloric acid solution, glacial acetic acid solution, and citric acid solution; in this embodiment, the acid solution is glacial acetic acid solution;

[0096] 8) ultrafiltration of the recombinant collagen eluate after treatment in step 7) until the conductivity is less than 0.6 mS / cm;

[0097] 9) The pH of the treated solution in step 8) is adjusted to 8.5-9, and then washed with sodium hydroxide and loaded onto an anion exchange chromatography column equilibrated with injection water to collect the flow-through solution.

[0098] 10) Adding protease to the flow-through liquid obtained after the treatment in step 9) to obtain the recombinant collagen liquid.

[0099] Example 2-22

[0100] Examples 2-22 provide a recombinant collagen and a recombinant collagen solution, respectively.

[0101] The difference between the above embodiment and embodiment 1 is that the copy numbers of each type of collagen are different, as shown in Table 1. The remaining parameters and steps are the same as the method and parameters provided in embodiment 1.

[0102] The differences between the above embodiments 1-22 are as follows:

[0103] The difference between Examples 1-4 is that the copy numbers of type I collagen are different.

[0104] The difference between Example 5-7 and Example 3 is that the copy numbers of type II collagen are different.

[0105] The difference between Examples 8-11 and Example 6 is that the copy numbers of type III collagen are different.

[0106] The difference between Examples 12-15 and Example 6 is that the copy numbers of type IV collagen are different.

[0107] The difference between Examples 16-19 and Example 13 is that the copy numbers of type XVII collagen are different.

[0108] The difference between Examples 20-22 and Example 17 is that the protease and linker sequence are different.

[0109] Comparative Example

[0110] Comparative Examples 1-5

[0111] Comparative Examples 1-5 each provide a collagen protein. The difference between them and Example 13 is that they only include one collagen protein. The remaining parameters and steps are the same as the method and parameters provided in Example 13.

[0112] The differences between Comparative Examples 1-5 and Example 13 are as follows:

[0113] Comparative Example 1: The type of collagen is type I collagen, and the sequence includes the amino acid sequence shown in SEQ ID NO.1.

[0114] Comparative Example 2: The type of collagen is type II collagen, and the sequence includes the amino acid sequence shown in SEQ ID NO.2.

[0115] Comparative Example 3: The type of collagen is type III collagen, and the sequence includes the amino acid sequence shown in SEQ ID NO.3.

[0116] Comparative Example 4: The type of collagen is type IV collagen, and the sequence includes the amino acid sequence shown in SEQ ID NO.4.

[0117] Comparative Example 5: The type of collagen is type XVII collagen, and the sequence includes the amino acid sequence shown in SEQ ID NO.5.

[0118] Comparative Example 6

[0119] Comparative Example 6 provides a recombinant collagen solution. The difference between it and Example 17 is that: each type of collagen solution is prepared separately, and finally mixed in proportion to prepare the recombinant collagen solution, and the collagen solution used is the collagen solution prepared by Comparative Examples 1-5. The remaining parameters and steps are the same as the method and parameters provided in Example 17.

[0120] Comparative Examples 7-11

[0121] Comparative Examples 7-11 provide a recombinant collagen and a recombinant collagen solution, respectively.

[0122] The difference between the comparative example and Example 17 is that the sequences of the various types of collagen are different. The remaining parameters and steps are the same as those provided in Example 17.

[0123] The sequences of various types of collagen in the above comparative examples are as follows:

[0124] In Comparative Example 7, the type I collagen sequence is different from that in Example 17. The type I collagen sequence in this comparative example includes the amino acid sequence shown in SEQ ID NO.11.

[0125] In Comparative Example 8, the type II collagen sequence is different from that in Example 17. The type II collagen sequence in this comparative example includes the amino acid sequence shown in SEQ ID NO.12.

[0126] In Comparative Example 9, the type III collagen is different from that in Example 17. The type III collagen sequence in this comparative example includes the amino acid sequence shown in SEQ ID NO.13.

[0127] In Comparative Example 10, the type IV collagen sequence is different from that in Example 17. The type IV collagen sequence in this comparative example includes the amino acid sequence shown in SEQ ID NO.14.

[0128] In Comparative Example 11, the type XVII collagen sequence is different from that in Example 17. The type XVII collagen sequence in this comparative example includes the amino acid sequence shown in SEQ ID NO.15.

[0129] Performance testing

[0130] The following tests were performed on the recombinant collagen solutions provided in the above examples and comparative examples, respectively.

[0131] (I) Expression levels and impurity detection of various types of collagen

[0132] The expression of collagen in the recombinant collagen solution provided in the above embodiment and comparative example was detected by WB (enhanced version of SDS-PAGE) and BCA quantitative method. The following data were obtained by calculation using a standard as a control. The impurities in the recombinant collagen solution provided in the above embodiment and comparative example were detected by ultraviolet absorption method, and the following data were obtained by calculation. The above test results are shown in Table 2.

[0133] Table 2 Test results

[0134]

[0135] According to the test results in Table 2, compared with the test results of the comparative example, it can be seen that the impurity content in the recombinant collagen liquid provided by the present application is lower.

[0136] (II) Testing of the moisturizing, firming, anti-wrinkle and repairing effects of recombinant collagen solution

[0137] (1) Moisturizing efficacy test

[0138] Test purpose and principle: The water in the stratum corneum of the skin plays an important role in maintaining skin health, preventing skin aging and moisturizing. The reason for dry skin is insufficient water in the stratum corneum, and the water is often combined with the stratum corneum in the form of bound water. An ideal moisturizing product should have the ability to maintain moisture, that is, the smaller the change in moisture, the stronger the moisturizing ability. This test method continuously monitors the change in moisture weight of the treated test sample and plots a moisture change rate △m-time change curve of the test sample. The moisturizing ability of the test sample is observed according to the trend of the moisture change rate △m curve, and then the moisturizing effect of the test sample on the skin is evaluated.

[0139] Correlation of test methods: This method is an in vitro method. The relevant literature shows that the trend of the in vivo method is basically consistent with the results of this in vitro method, and the two methods have a certain correlation. Through the experimental design, the test sample is compared with the positive control sample with moisturizing ability. If the moisture change rate △m curve of the test sample is less than (better than) the moisture change rate △m curve of the positive control, it can be considered that the test sample has a certain moisturizing ability.

[0140] Test index: moisture change rate △m.

[0141] Judgment criteria: Compare whether the moisture change rate △m curve of the test sample is less than (better than) the moisture change rate △m curve of the positive control to determine whether the sample has moisturizing effect.

[0142] Test method: weigh the sample; pre-treat the test sample; select a reagent with known hygroscopicity as the positive control; place the sample and the positive control in a constant temperature and humidity device at 37°C; weigh the test sample regularly; record the data, calculate the moisture change rate △m, and make a curve. The positive control reagent for this test is: DL-pyroglutamic acid.

[0143] The moisture change rate △M of the positive control at different times is shown in Table 3. The moisture change rate △m of the test sample within 48h.

[0144] Table 3 △m of the moisture change rate of the positive control at different times

[0145] Serial number Detection time (h) Positive control △m 1 2 0.2574 2 4 0.2550 3 6 0.2426 4 8 0.2411 5 24 1.2263 6 48 1.5070 7 72 1.0998

[0146] (2) Firming effect test

[0147] Purpose and principle of the test: Elastic fibers are composed of elastin and microfibrils, distributed in the dermis and subcutaneous tissue, making the skin elastic. However, due to the effects of some environmental factors such as ultraviolet radiation, pressure, pollution, etc., the production of elastase in the body will be promoted. Elastase is a member of the chymosin family, which will degrade elastin and cause the loss of connective tissue in the epidermis, thus leading to skin aging, wrinkles and photoaging.

[0148] Correlation of test methods: This method is an in vitro method and is applicable to cosmetics claiming firming effects. Relevant literature shows that the trend of the in vivo method is basically consistent with the results of this in vitro method, and the two methods have a certain correlation. Through the experimental design, the test samples and the blank control group were compared for the results of the elastase inhibition rate test. If the inhibition rate of the test sample is better (higher) than that of the blank control group and the statistical difference P value is <0.05, it can be considered that the test sample has a certain firming effect.

[0149] Test indicator: elastase inhibition rate.

[0150] Judgment criteria: Calculate the inhibition rate of elastase before and after adding the test substance, and the statistical difference P value is <0.054.

[0151] Test method: Weigh the sample; pre-treat the test substance to prepare a mixed solution; incubate at 25°C for 12 hours, then add an equal amount of elastase to the control group and continue incubation; test the OD value on the machine; record the data, calculate the inhibition rate and statistical difference P value.

[0152] The average inhibition rate of elastase in the blank control group was 0.49%.

[0153] (3) Anti-wrinkle efficacy testing

[0154] Purpose and principle of the test: External and body-specific factors can lead to the generation of free radicals. Free radicals have extremely strong oxidizing ability. They attack any biological molecules through oxidation reactions, and undergo various oxidation reactions with macromolecules such as lipids, sugars, proteins, and deoxyribonucleic acid, causing oxidative damage such as denaturation, cross-linking, and breakage, which in turn leads to the destruction of cell structure and function, as well as damage to body tissues and pathological changes in organs, which manifest as aging and wrinkles on the skin. When the test sample is in the presence of a free radical scavenger, the light absorption of the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) reagent (a stable, long-lived free radical) is weakened. This can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity.

[0155] Relevance of the test method: This method is an in vitro method and is applicable to cosmetics that claim anti-aging, anti-wrinkle, and antioxidant activity, as shown in relevant literature. When a free radical scavenger is present in the test sample, the degree of fading of the DPPH solution is linearly related to the free radical scavenging ability of the free radical scavenger, which can be used to evaluate the free radical scavenging ability of the test sample, that is, the size of the antioxidant activity. Through experimental design, the DPPH free radical scavenging rate test results of the test sample and the blank control group are compared. If the scavenging rate of the test sample is better (higher) than the scavenging rate of the blank control group and the statistical difference P value is <0.05, it can be considered that the test sample has anti-wrinkle effect.

[0156] Test indicator: DPPH free radical scavenging rate.

[0157] Judgment criteria: Calculate the DPPH free radical scavenging rate before and after adding the test substance, and the statistical difference P value is <0.05.

[0158] Test method: Weigh the sample; pre-treat the test sample and prepare a solution for use; add the free radical DPPH, prepare multiple tubes of samples, shake well, and set aside; after standing at room temperature for 10 minutes, test the absorbance on the machine; record the data, calculate the clearance rate and statistical difference P value before and after adding the test substance.

[0159] The average DPPH free radical scavenging rate of the blank control group was 0.35%.

[0160] (4) Short-term repair efficacy testing

[0161] Testing method: Select healthy Chinese male / female subjects aged 18-60 years old. Use the self-before and after control and blank control method. Before using the product (denoted as T0h ), 1 hour after using the product (recorded as T 1h ) measure the TEWL value of the test area skin respectively; the evaluation results before and after product use are analyzed and compared by statistical test methods to determine whether there are statistical differences.

[0162] Testing environment: Testing environment temperature: 20-22℃; relative humidity: 40-60%, in line with the design requirements.

[0163] Testing indicators and methods:

[0164] Index name: Transepidermal water loss TEWL value;

[0165] Test instrument / required reagents: AS-Tewl transepidermal moisture loss tester;

[0166] Measurement area: inner forearm skin;

[0167] Measuring time: T 0h , T 1h ;

[0168] Test method: Use AS-TEWL transepidermal water loss tester to measure the transepidermal water loss rate TEWL value of the test area.

[0169] The formula for calculating the rate of change is as follows:

[0170] Control difference = T 1h Control-T 0h Control;

[0171] Sample difference = T 1h Sample-T 0h sample;

[0172] Change rate of control area before and after product use = control difference / T 0h Control*100%;

[0173] Sample area change rate before and after product use = sample difference / T 0h Sample *100%.

[0174] In the formula, T 0h Control – initial values ​​of skin parameters in the control area before the product is used in the test area;

[0175] T 1h Control: Skin parameter values ​​of the control area one hour after the product is used on the test area;

[0176] T 0h Sample – initial value of skin parameters before using the product on the test area;

[0177] T 1hSample - Skin parameter values ​​of the test area one hour after using the product.

[0178] Sample completion status: 30 subjects were enrolled and 30 subjects completed the experiment. The age ranged from 19 to 41 years old, with an average age of 25.10±0.72 years old. The product was used for 1 hour of transdermal 3D collagen.

[0179] Product usage and tolerance feedback: According to the "Technical Specifications for Safety of Cosmetics" (2015 edition), each subject used the product transdermal 3D collagen for 1 hour, and the adverse reaction record results showed that no adverse reactions occurred.

[0180] The TEWL value of the control area of ​​the subject decreased significantly compared with the initial value, with a decrease rate of 9.21%. The decrease in the TEWL value of the skin indicates that the amount of water lost by the skin is reduced.

[0181] The above test results are shown in Table 4.

[0182] Table 4 Test results of moisturizing, firming, anti-wrinkle and repairing effects of recombinant collagen solution

[0183]

[0184]

[0185] According to the test results in Table 4, by comparing the test results of Example 17 with those of Comparative Examples 6-11, it can be seen that the recombinant collagen solution provided by the present application has good moisturizing, firming, anti-wrinkle and repairing effects.

[0186] By comparing the test results of Examples 1-19, it can be seen that the present application controls the copy number ratio of type I collagen, type II collagen, type III collagen, type IV collagen and type XVII collagen in the recombinant collagen solution to m:n:o:p:q=(6-14):(2-6):(2-10):(1-6):(1-6), which can effectively improve the moisturizing, firming, anti-wrinkle and repair effects of the recombinant collagen solution provided by the present application. Further, the present application controls the copy number ratio of type I collagen, type II collagen, type III collagen, type IV collagen and type XVII collagen in the recombinant collagen solution to m:n:o:p:q=(8-10):(3-4):(4-8):(2-3):(2-3), which can further improve the moisturizing, firming, anti-wrinkle and repair effects of the recombinant collagen solution provided by the present application.

[0187] By comparing the test results of Example 17 and Examples 20-22, it can be seen that the difference in protease recognition sequences has a certain impact on the final test results. By comparing the test results, type I collagen, type II collagen, type III collagen, type IV collagen, type XVII collagen and the number of copies of each type of collagen are preferably connected by a sequence that can be recognized and cleaved by TEV protease.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A transdermal recombinant multi-type collagen, characterized in that: The recombinant collagen consists of type I collagen, type II collagen, type III collagen, type IV collagen and type XVII collagen; The type I collagen is an amino acid sequence as shown in SEQ ID NO. 1, and the copy number is m, m≥1; The type II collagen is an amino acid sequence as shown in SEQ ID NO. 2, and the copy number is n, n≥1; The type III collagen is an amino acid sequence as shown in SEQ ID NO. 3, and the copy number is o, o≥1; The type IV collagen is an amino acid sequence as shown in SEQ ID NO. 4, and the copy number is p, p≥1; The type XVII collagen is an amino acid sequence as shown in SEQ ID NO. 5, and the copy number is q, q≥1; The copy numbers of the type I collagen, the type II collagen, the type III collagen, the type IV collagen and the type XVII collagen are: m is 6-14; n is 2-6; o is 2-10; p is 1-6; q is 1-6; The type I collagen, the type II collagen, the type III collagen, the type IV collagen, the type XVII collagen, and the copies of each type of collagen are connected by sequences that can be recognized and cut by proteases; The protease is selected from TEV protease, EK protease, collagenase or Thrombin thrombin; The N-terminus or C-terminus of the recombinant collagen is connected to a transdermal short peptide via a linker sequence; the transdermal short peptide is an amino acid sequence as shown in SEQ ID NO.

6.

2. The transdermal multi-type recombinant collagen according to claim 1, characterized in that: The copy numbers of the type I collagen, the type II collagen, the type III collagen, the type IV collagen and the type XVII collagen are: m is 8-10; n is 3-4; o is 4-8; p is 2-3; q is 2-3.

3. The transdermal multi-type recombinant collagen according to claim 1, characterized in that: The recombinant collagen has an amino acid sequence as shown in SEQ ID NO. 7-SEQ ID NO.

10.

4. The transdermal multi-type recombinant collagen according to claim 1, characterized in that: The protease is TEV protease.

5. A gene encoding the transdermal multi-type recombinant collagen according to any one of claims 1 to 4.

6. A method for preparing a recombinant collagen solution, characterized in that: The preparation method specifically comprises the following steps: designing a corresponding gene sequence for the transdermal multi-type recombinant collagen described in any one of claims 1 to 4, constructing a recombinant vector comprising the gene sequence, and introducing the gene sequence into competent cells to screen for a strain with high expression of the multi-type recombinant collagen; using the strain with high expression to prepare the multi-type recombinant collagen, and purifying to obtain a recombinant collagen solution.

7. A recombinant collagen solution prepared by the preparation method according to claim 6.

8. Use of the recombinant collagen solution according to claim 7 in preparing a composition for tissue repair, scar healing, cell repair, anti-aging and skin moisturizing.

Images