Recombinant TAT transmembrane collagen as well as preparation and application methods thereof
By fusing TAT membrane-penetrating peptides to the C-terminus of collagen, recombinant TAT membrane-penetrating collagen was prepared, solving the problem of difficult collagen absorption and achieving highly efficient skin absorption.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-04-07
AI Technical Summary
Existing collagen molecules are large, making them difficult to absorb. Direct application is not effective; transdermal injection is required for it to be effective.
The transmembrane peptide TAT was fused to the C-terminus of collagen to form recombinant TAT transmembrane collagen, which was then prepared and purified using a Pichia pastoris expression system.
Recombinant TAT transmembrane collagen significantly improves skin absorption rate, with an 8-hour absorption rate twice that of pure collagen, and its effects are remarkable when applied directly to the skin.
Smart Images
Figure CN121800942A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant TAT transmembrane collagen, its preparation method, composition, and application. Background Technology
[0002] Collagen is the most abundant protein in the human body, accounting for approximately 25%-33% of total body protein and up to 70% of the dry matter in skin. It is widely distributed in tissues such as skin, bones, tendons, ligaments, and cartilage, and is a major component of the extracellular matrix, providing mechanical support, maintaining structural stability and elasticity. The demand for collagen continues to grow in the fields of aesthetic medicine, skincare, medical treatment, and health care. In aesthetic medicine, collagen injections have become a popular filler material; in skincare, recombinant collagen is favored for its highly effective repair and anti-aging effects; and in the medical field, collagen is used for wound repair, tissue engineering, and cardiovascular stent coatings.
[0003] However, due to its large molecular weight, collagen is difficult to absorb, and direct application yields minimal results. Transdermal injection is often required to achieve good effects. Therefore, there is an urgent need to invent a recombinant collagen that can be directly absorbed through the skin to solve the current difficulties in the absorption of collagen through application. Summary of the Invention
[0004] To address the technical problems existing in the prior art, the present invention provides the following technical solution:
[0005] On one hand, a recombinant TAT transmembrane collagen is provided, wherein the recombinant TAT transmembrane collagen is a fusion protein in which the transmembrane peptide TAT is fused to the C-terminus of collagen, and the amino acid sequence of the recombinant TAT transmembrane collagen is shown in SEQ ID NO:1.
[0006] On the other hand, an isolated nucleic acid molecule encoding the recombinant TAT transmembrane collagen is provided, the nucleotide sequence of which is shown in SEQ ID NO:2.
[0007] On the other hand, a recombinant expression vector is also provided, which contains the nucleic acid molecules described above.
[0008] Preferably, the recombinant expression vector is pPIC9K.
[0009] On the other hand, a recombinant host cell is also provided, which contains the recombinant expression vector described above, or the genome of the recombinant host cell is integrated with the nucleic acid molecules described above.
[0010] Preferably, the host cell is Pichia pastoris cell GS115.
[0011] On the other hand, a method for constructing the recombinant host cell described above is also provided, comprising the following steps:
[0012] (1) The nucleic acid molecule encoding the recombinant TAT transmembrane collagen was cloned into a eukaryotic expression vector to obtain a recombinant plasmid;
[0013] (2) The recombinant plasmid is introduced into a host cell to obtain a transformant;
[0014] (3) Screening for transformants that can express the recombinant TAT transmembrane collagen, thus obtaining the transformants.
[0015] On the other hand, a method for preparing the above-mentioned recombinant TAT transmembrane collagen is also provided, comprising the steps of culturing the above-mentioned recombinant host cells and isolating and purifying the recombinant TAT transmembrane collagen from the culture.
[0016] On the other hand, a composition is also provided comprising the recombinant TAT transmembrane collagen described above, and a pharmaceutically or cosmetically acceptable carrier.
[0017] On the other hand, the use of the above-described recombinant TAT transmembrane collagen or the composition thereof in the preparation of cosmetics or medical devices for transdermal drug delivery is also provided.
[0018] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:
[0019] This invention provides a method for promoting the absorption of recombinant collagen and its applications. The method primarily involves fusing a TAT membrane-penetrating peptide to the C-terminus of recombinant collagen, forming recombinant TAT membrane-penetrating collagen. Recombinant TAT membrane-penetrating collagen possesses excellent hydrophilicity and high activity, thus showing potential applications in biomedical materials, cosmetics, and skincare products. The fusion of the membrane-penetrating TAT structure at the C-terminus of recombinant type III collagen promotes collagen absorption, significantly enhancing skin absorption. Compared to pure recombinant collagen, TAT membrane-penetrating collagen exhibits more than twice the absorption rate after 8 hours, indicating that direct application of TAT membrane-penetrating collagen to the skin results in better absorption and a more pronounced effect. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1This is a schematic diagram of the COLⅢ-TAT gene structure provided in an embodiment of the present invention;
[0022] Figure 2 This is a schematic diagram of optimized gene sequence detection provided in an embodiment of the present invention;
[0023] Figure 3 This is a graph showing the effect of gel running verification provided in Embodiment 3 of the present invention;
[0024] Figure 4 This is a statistical bar chart of the relative proliferation rate of recombinant TAT transmembrane collagen cells provided in Example 5 of the present invention;
[0025] Figure 5 This is a bar chart of cell adhesion rate obtained from a recombinant TAT transmembrane collagen adhesion experiment provided in Example 6 of the present invention;
[0026] Figure 6 This is a Raman image of a transdermal experiment of recombinant TAT transmembrane collagen provided in Embodiment 7 of the present invention;
[0027] Figure 7 This is a Raman image of another recombinant TAT transdermal collagen transdermal experiment provided in Example 7 of the present invention. Detailed Implementation
[0028] The technical solution of the present invention will now be described with reference to the accompanying drawings.
[0029] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.
[0030] In the embodiments of this invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning. Similarly, the terms "of," "corresponding (relevant)," and "corresponding" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning.
[0031] In this embodiment of the invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meaning they express is the same.
[0032] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.
[0033] The reagents and equipment used in this embodiment can be purchased by the user or provided by the laboratory.
[0034] This invention provides a method for promoting the absorption of recombinant collagen and its application, which mainly involves fusing a TAT membrane-penetrating peptide to the C-terminus of recombinant collagen to form recombinant TAT membrane-penetrating collagen.
[0035] The application principles and experimental conclusions of the present invention will be described below with reference to various embodiments.
[0036] Example 1: Construction of a Pichia pastoris expression system containing recombinant TAT transmembrane collagen
[0037] Using pPIC9K (purchased from Invitrogen) as the backbone, optimized gene sequences were introduced into the multiple cloning site to obtain pPIC9K-COLⅢ-TAT, which was then transformed into Pichia pastoris GS115. The detailed steps are as follows:
[0038] 1. Based on the mature peptide sequences of type III collagen published in the UniProt protein resource database (https: / / www.uniprot.org / ), a short peptide sequence (as shown in SEQ ID NO:5) was selected, and the transmembrane TAT sequence was designed at the C-terminus of the short peptide sequence, i.e., the amino acid sequence of recombinant TAT transmembrane collagen is shown in SEQ ID NO:1:
[0039] 2. Gene sequences were reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ) to select the preferred codons required for expression in the host Pichia pastoris. During the design process, the Xhol and NotI restriction sites were removed. GenScript Biotech Co., Ltd. was commissioned to synthesize the optimized COLⅢ-TAT gene. The optimized gene sequence is shown in SEQ ID NO:2.
[0040] 3. Construction of recombinant strain: The target gene shown in SEQ ID NO.2 ( Figure 1 (COLIII represents the inserted gene). Xhol and NotI double restriction sites were introduced and inserted into the expression vector pPIC9K to obtain the recombinant plasmid (see schematic diagram). Figure 1 The recombinant plasmids were linearized and electroporated into Pichia pastoris GS115 cells. Colony PCR identification was performed, and the cells were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing (see schematic diagram). Figure 2(COLIII-TAT represents the recombinant TAT transmembrane collagen gene sequence) Primer F and R base sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
[0041] Example 2: Screening of Pichia pastoris expression strains containing recombinant TAT transmembrane collagen. The recombinants were plated onto YPD solid plates containing G418 at concentrations of 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml, respectively, and incubated at 30°C for 2-3 days. The growth status of the recombinants was observed.
[0042] Example 3: Preliminary expression of Pichia pastoris containing recombinant TAT transmembrane collagen. 50 μl of the identified positive strains (1#, 2#, 3#) were inoculated into a conical flask containing 10 ml of BMGY and cultured overnight at 30°C and 220 rpm. The culture was then shaken until OD600 = 2–6 (logarithmic growth, approximately 16–18 h). Cells were collected by centrifugation at 5000 rpm for 5 min at room temperature, the supernatant was removed, and the cells were resuspended in 10 ml of BMGY for induction. Every 24 h, 1 ml of the culture medium was sampled and methanol was added to a final concentration of 0.5% for further induction. At the following time points (0, 24, 48, 72, 96 h), samples were centrifuged at 10000 rpm for 2 min to collect the supernatant, which was then subjected to 96 h SDS-PAGE verification (see schematic diagram). Figure 3 ).
[0043] Example 4: Purification of recombinant TAT transmembrane collagen product
[0044] The fermentation broth of strain #1 in Example 3 was centrifuged, and the supernatant was collected. The supernatant was filtered through a filter membrane, purified by gel column chromatography, and then freeze-dried to obtain the final product. The specific purification steps for the above-mentioned human recombinant collagen are as follows: The fermentation broth was centrifuged at 4200 rpm for 30 min, and the supernatant was collected. The supernatant was concentrated and washed with an ultrafiltration membrane to remove salts and pigments. An appropriate amount of the above collagen solution was taken and purified by SP resin column chromatography. The eluent containing collagen was collected, and the eluent was washed, desalted, concentrated, and replaced with an ultrafiltration membrane. The target protein was collected by freeze-drying.
[0045] Example 5: Relative proliferation rate of recombinant TAT transmembrane collagen cells
[0046] HaCat cells (human keratinocytes) in the logarithmic growth phase were taken at a dose of 1×10⁻⁶. 5HaCat cells were seeded at a density of 100 µL / well in 96-well plates, divided into control and experimental groups. The cells were incubated in a CO2 cell culture incubator at 37°C and 5% CO2 for 24 h. A serum-free culture medium was used to prepare a 0.5 mg / ml solution of the recombinant TAT transmembrane collagen obtained in Example 4, and the solution was sterilized by filtration through a 0.22 µm filter. The competitor protein was also prepared at a concentration of 0.5 mg / ml and sterilized by filtration through a 0.22 µm filter. After 24 h of routine HaCat cell culture, the old culture medium was discarded, and 100 μL of serum-free culture medium or 100 μL of competitor collagen was added. The control group received an equal volume of serum-free culture medium, while the experimental group received 100 μL of the recombinant TAT transmembrane collagen solution. Each group had three replicates. After culturing for another 24 hours, the culture medium was discarded, and 100 μL of CCK-8 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) diluted 10-fold with serum-free culture medium was added to each well. The cells were then incubated in a cell culture incubator for another 2 hours. The relative cell proliferation rate was detected using the CCK-8 assay, with absorbance measured at 450 nm using a microplate reader. Cell proliferation rate (RGR)% was calculated as: (Experimental group absorbance value / Normal control group absorbance value) × 100% (e.g., ...). Figure 4 (As shown) Within the selected concentration range, it exhibits a certain cell proliferation ability and no cytotoxicity.
[0047] Example 6: Adhesion experiment of recombinant TAT transmembrane collagen
[0048] HaCat cells (human keratinocytes) in the logarithmic growth phase were taken at a dose of 1×10⁻⁶. 5 Recombinant TAT transmembrane collagen was seeded at a density of 100 µL / well in 96-well plates, divided into control and experimental groups. The plates were incubated in a CO2 cell culture incubator at 37°C and 5% CO2 for 24 h. A serum-free culture medium was used to prepare a 0.5 mg / ml solution of the recombinant TAT transmembrane collagen obtained in Example 4, and the solution was sterilized by filtration through a 0.22 µm filter. Competitor collagen was also prepared at a concentration of 0.5 mg / ml, and the solution was sterilized by filtration through a 0.22 µm filter.
[0049] HaCat cells were cultured for 24 h using standard methods. The old culture medium was discarded, and 100 μL of serum-free culture medium or 100 μL of a competitor's protein solution was added. The control group received an equal volume of serum-free culture medium, while the experimental group received 100 μL of recombinant TAT transmembrane collagen sample solution. Each group had three replicates. After another 24 h of culture, the culture medium was discarded, and 100 μL of CCK-8 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) diluted 10-fold with serum-free culture medium was added to each well. The cells were then incubated in a cell culture incubator for 2 h, and the absorbance was measured at 450 nm using a microplate reader. The relative cell adhesion rate was thus determined (see schematic diagram). Figure 5This is sufficient. The cell adhesion rate reflects the activity of collagen. The higher the protein activity, the better it can provide a high-quality external environment for cells in a short time, helping cells adhere. The cell adhesion rate reflects the activity of collagen. With the adhesion rate of the control group as 1, the relative cell adhesion activity of recombinant TAT transmembrane collagen can be calculated.
[0050] Example 7: Transdermal Experiment of Recombinant TAT Permeabilized Collagen
[0051] Raman spectroscopy was performed on isolated porcine skin. The samples used for testing were recombinant TAT transmembrane collagen and collagen, and a 1×1 cm sample was selected. 2 The test skin model was used as the test area, with a total of 6 equally sized regions selected. Five points were taken from each region using both vertex and center-based methods. The average of three scans for each point was considered a valid data point. Five points within each test area were selected for parallel testing three times, and in vitro porcine skin Raman spectroscopy was performed at time points of 0 h, 2 h, 4 h, 6 h, and 8 h. The recombinant TAT permeabilized collagen and the relative permeability of collagen in the sample were calculated.
[0052] Through the Figure 6 Depth analysis of Raman images can reveal the distribution of collagen in recombinant TAT transmembrane collagen at different depths in isolated porcine skin, as shown in the figure. Figure 6-7 As shown, the relative permeability of collagen on isolated pig skin at 0 h, 2 h, 4 h, 6 h and 8 h after using the sample recombinant TAT transmembrane collagen was 0%, 2.54%, 4.02%, 5.88% and 12.47%, respectively.
[0053] Through the Figure 7 Depth analysis of Raman images can reveal the distribution of collagen in the sample at different depths in isolated pig skin, as shown in the following diagram. Figure 7 As shown, the relative permeability of collagen on isolated pig skin at 0 h, 2 h, 4 h, 6 h and 8 h after application of sample collagen was 0%, 3.86%, 4.11%, 5.20% and 8.39%, respectively.
[0054] Further analysis of the relative permeability revealed that the relative permeability of collagen in the recombinant TAT transmembrane collagen sample at 6 h and 8 h was 1.13 times and 1.49 times that of the collagen in the sample collagen, respectively.
[0055] Therefore, the recombinant TAT transmembrane collagen provided by this invention possesses excellent hydrophilicity and high activity, thus showing potential applications in biomedical materials, cosmetics, and skincare products. The C-terminus of the recombinant type III collagen fused with the transmembrane structure TAT promotes collagen absorption and significantly enhances skin absorption.
[0056] Compared to simple recombinant collagen, TAT transmembrane collagen has more than twice the absorption rate in 8 hours, indicating that TAT transmembrane collagen can be better absorbed and has a more obvious effect when applied directly to the skin.
[0057] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A recombinant TAT transmembrane collagen, characterized in that, The recombinant TAT transmembrane collagen is a fusion protein in which the transmembrane peptide TAT is fused to the C-terminus of collagen, and the amino acid sequence of the recombinant TAT transmembrane collagen is shown in SEQ ID NO:
1.
2. An isolated nucleic acid molecule encoding the recombinant TAT transmembrane collagen of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:
2.
3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2.
4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is pPIC9K.
5. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant expression vector of claim 3 or 4, or the genome of the recombinant host cell integrates the nucleic acid molecule of claim 2.
6. The recombinant host cell according to claim 5, characterized in that, The host cell was Pichia pastoris cell GS115.
7. A method for constructing the recombinant host cell according to any one of claims 5-6, characterized in that, Includes the following steps: (1) The nucleic acid molecule encoding the recombinant TAT transmembrane collagen was cloned into a eukaryotic expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid is introduced into a host cell to obtain a transformant; (3) Screening for transformants that can express the recombinant TAT transmembrane collagen, thus obtaining the transformants.
8. A method for preparing the recombinant TAT transmembrane collagen of claim 1, characterized in that, The method includes the steps of culturing the recombinant host cells according to any one of claims 5-6 and isolating and purifying the recombinant TAT transmembrane collagen from the culture.
9. A composition, characterized in that, It comprises the recombinant TAT transmembrane collagen as described in claim 1, and a pharmaceutically or cosmetically acceptable carrier.
10. Use of the recombinant TAT transmembrane collagen of claim 1 or the composition of claim 9 in the preparation of cosmetics or medical devices for transdermal drug delivery.