Novel recombinant protein TAT-FLG2 with cell membrane penetrating capacity and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a novel recombinant protein TAT-FLG2 with cell membrane penetrating capacity and application. The novel recombinant protein TAT-FLG2 provided by the invention comprises a part of peptide fragment of silk protein 2 and a section of cell-penetrating peptide which are connected through a section of flexible connexon. The three-dimensional structure of the novel recombinant protein TAT-FLG2 has a shape similar to a V shape. The novel recombinant protein TAT-FLG2 provided by the invention has the capability of efficiently penetrating cell membranes and the capability of promoting the migration of fibroblasts.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel recombinant protein TAT-FLG2 with cell membrane penetration ability and application thereof. Background Art

[0002] Filaggrin (FLG) is an important molecule that connects keratin fibers in the stratum corneum of human skin. With the help of FLG monomers, keratin fibers can be regularly aggregated to form a solid physical barrier in the outermost layer of the epidermis, thereby preventing the loss of epidermal moisture and the invasion of external allergic substances. As one of the important subtypes of filaggrin, filaggrin 2 (FLG2) is synthesized in keratinocytes and can be distributed in many parts. It is gradually degraded by enzymes during the migration of keratinocytes. FLG2 degraded by enzymes will become small molecules required by the stratum corneum (such as natural moisturizing factor, etc.), effectively maintaining a certain level of moisture in the skin and promoting fibroblasts to maintain good proliferation and metabolic capacity. At the same time, the degradation products of FLG2 also contain more histidine, which can generate pyrrolidone-5-carboxylic acid, trans-uridylic acid, etc. after further metabolism, which can effectively maintain the weak acidic environment of the skin and resist microbial invasion. However, direct external application of FLG2 has the problem of difficulty in penetrating stratum corneum cells and thus difficulty in entering the dermis to effectively function. In the prior art, there are strategies for applying other small molecules to promote the increase of the content or expression of filaggrin in the epidermis from the side. For example, the patent with publication number CN116098834A and invention name "Application of an oat β-glucan hydrolyzate in skin care products" discloses that a skin care product containing oat β-glucan hydrolyzate is used to increase the expression of filaggrin in the epidermis. For another example, the patent with publication number "CN115944581A" and invention name "A rose cell fluid and its preparation method and application" discloses that a skin topical preparation containing rose polysaccharides can increase the content of filaggrin. At present, there are few studies on FLG2 or preparations containing FLG2 that have high penetration ability or efficiency when directly applied into the skin.

[0003] TAT (AYGRKKRRQRRR) is derived from the transactivator of transcription of human immunodeficiency virus HIV-1 and is a cell-penetrating peptide that has the ability to efficiently deliver various bioactive substances into cells. So far, there has been no report on the application of TAT to filaggrin to improve the membrane permeability of filaggrin.

[0004] In summary, it is necessary to propose a recombinant filaggrin with strong cell penetrating ability to alleviate or partially alleviate the deficiencies of the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a novel recombinant protein TAT-FLG2 with cell membrane penetration ability and its application. The present invention specifically adopts the following technical scheme.

[0006] One aspect of the present invention provides a novel recombinant protein.

[0007] A novel recombinant protein TAT-FLG2, comprising a partial peptide segment of filaggrin 2 and a cell-penetrating peptide; the amino acid sequence of the novel recombinant protein TAT-FLG2 is shown in SEQ ID NO.1.

[0008] Furthermore, the nucleotide sequence of the novel recombinant protein TAT-FLG2 is shown in SEQ ID NO.2.

[0009] Furthermore, the cell-penetrating peptide is YGRKKRRQRRR.

[0010] Furthermore, the partial peptide segment of filaggrin 2 and the cell-penetrating peptide are connected via a flexible linker; the flexible linker is GGGGSGGGGSGGGGS.

[0011] Furthermore, the three-dimensional structure of the novel recombinant protein TAT-FLG2 has a V-like shape.

[0012] Another aspect of the present invention provides the application of the novel recombinant protein.

[0013] A recombinant expression vector comprises the gene of the novel recombinant protein TAT-FLG2.

[0014] In some optional embodiments, the type of the vector includes a plasmid vector, a phage, an adenovirus vector or a baculovirus vector.

[0015] A recombinant bacterium expressing the novel recombinant protein TAT-FLG2.

[0016] In some alternative embodiments, the bacteria include Escherichia coli, Bacillus subtilis or Pichia pastoris strains.

[0017] The application of the novel recombinant protein TAT-FLG2 in the preparation of topical skin preparations.

[0018] Furthermore, the skin external preparation includes a spray, an aqueous solution, a gel or an emulsion.

[0019] The new recombinant protein TAT-FLG2 contained in the above-mentioned preparation has high cell membrane penetration ability and can pass through the skin barrier and enter the dermis to exert its effect when applied externally.

[0020] A spray for promoting wound healing, comprising the novel recombinant protein TAT-FLG2.

[0021] Furthermore, the spray may also contain other pharmaceutically acceptable adjuvants and / or auxiliary agents.

[0022] Optionally, a preparation for promoting wound healing may also be provided, wherein the preparation comprises the novel recombinant protein TAT-FLG2; the preparation may be in the form of an aqueous solution, a gel or an emulsion.

[0023] A spray for promoting fibroblast migration, comprising the novel recombinant protein TAT-FLG2.

[0024] The above-mentioned spray for promoting fibroblast migration can be used externally to accelerate wound healing, or used as a reagent for cell scratch experiments.

[0025] Beneficial technical effects: The present invention provides a novel recombinant protein TAT-FLG2, which has the ability to efficiently penetrate cell membranes. Compared with natural filaggrin 2, the recombinant protein TAT-FLG2 modified by the present invention has stronger cell penetration ability, so it can be prepared as a direct external preparation. In addition, the novel recombinant protein TAT-FLG2 of the present invention also has the ability to promote fibroblast migration and promote wound healing. Obviously, the novel recombinant protein TAT-FLG2 provided by the present invention has a good market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is the crystal structure diagram of the new recombinant protein TAT-FLG2 predicted by Alphafold 3; Figure 2 This is an SDS-PAGE image of the novel recombinant protein TAT-FLG2 purified in one of the embodiments of the present invention; Figure 3 This is a diagram showing the hydrophilicity analysis results of the novel recombinant protein TAT-FLG2 in one of the embodiments of the present invention; Figure 4 The results of the experiment on the effect of different concentrations of the novel recombinant protein TAT-FLG2 on the survival rate of human embryonic kidney cells (293T) and mouse epithelial fibroblasts (L929) in one of the embodiments of the present invention are as follows; Figure 5 This is the statistical result of the average fluorescence intensity after the novel recombinant protein TAT-FLG2 was co-cultured with vascular endothelial cells for a certain period of time in one of the embodiments of the present invention; Figure 6 This is the experimental result of using the novel recombinant protein TAT-FLG2 and natural filaggrin 2 to promote the scratch healing efficiency of L929 cells in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0030] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0031] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0032] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0033] Definition of noun: The "V-shaped" mentioned in the present invention means that the three-dimensional structure of the novel recombinant protein TAT-FLG2 of the present invention is V-shaped as a whole, with only slight deformation at the two ends.

[0034] Main materials and reagents: Human embryonic kidney cells 293T were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-502, animal species: human, tissue sources: fetus, embryonic kidney; mouse fibroblasts L929 were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-5039, animal species: mouse, tissue sources: subcutaneous connective tissue, areola and fat.

[0035] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0036] The pET-22b(+) plasmid is a commercially available E. coli expression vector purchased from Beijing Qingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin.

[0037] Restriction enzymes NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Bioengineering Co., Ltd. IPTG, ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solebow Technology Co., Ltd. DMEM culture medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd.

[0038] All reagents were used according to the reagent instructions.

[0039] Culture medium: Each liter of LB medium contains: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and the pH is adjusted to 7.0.

[0040] Preparation method: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950ml double distilled water, adjust the pH to 7.0 with sodium hydroxide solution, and dilute to 1L with double distilled water. If preparing solid culture medium, add agar at 1.5g / 100ml. Sterilize at 121℃ high pressure steam for 30min.

[0041] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0042] Example 1 This embodiment provides an example of TAT-FLG2 gene design and protein expression.

[0043] 1. Genetic Design The designed gene contains a cell-penetrating peptide (YGRKKRRQRRR) and a partial amino acid sequence of filaggrin 2. The protein expressed by the gene has 264 amino acids, and its sequence is shown in SEQ ID NO.1; the nucleotide sequence encoding the protein is shown in SEQ ID NO.2, and the total length is 792 bp. Further, NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) are added to the 5' end and 3' end of the gene, respectively, and the nucleotide sequence of the gene with restriction sites is shown in SEQ ID NO.3, and the total length is 801 bp. The sequences involved in this embodiment are shown in Table 1.

[0044] Table 1 Sequence information 2. Vector Construction The pET-22b(+) plasmid was used as an expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO.1) were subjected to double restriction digestion reaction with restriction endonucleases NdeI and XhoI, respectively, and then the target gene was connected to the pET-22b(+) vector by ligation reaction to obtain a ligation product. It is understood that other vectors, such as bacteriophage, adenovirus vector or baculovirus vector, can also be used.

[0045] 3. Conversion (1) Take out the competent E. coli BL21 (DE3) cells from the -80°C freezer and place them on ice for 5 min.

[0046] (2) After the glycerol containing the BL21(DE3) competent cells has melted, add the competent cells to the ligation product, pipette and release the pipette tip 3-4 times to mix, and place on ice for 30 min.

[0047] (3) Quickly wipe the tube wall dry with absorbent paper, then heat shock at 42°C for 90 seconds, and immediately place on ice for 2 minutes.

[0048] (4) Add 800 μL of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0049] (5) Collect the cells by centrifugation at 8000 rpm for 5 min, discard part of the supernatant, and use the remaining 100 μL of supernatant to resuspend the E. coli. Then, spread it evenly on LB solid culture medium containing 100 μg / mL ampicillin, place it in a 37°C incubator, and invert and culture for 10-16 h.

[0050] (6) Pick a single clone and inoculate it in a liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 10-16 hours, identify the positive clones and obtain the positive clones containing the target gene plasmid. It is understood that other bacteria can also be used for transformation, such as Bacillus subtilis or Pichia pastoris strains.

[0051] 4. Protein Expression and Purification (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench and cool it to room temperature. Add ampicillin to the LB medium in the clean bench and mix it to a final concentration of 100 μg / mL. Then, inoculate 200 μL / L of Escherichia coli (positive clone) containing the target gene plasmid into the LB medium. Place the LB medium in a shaker and culture it at a speed of 170 rpm and a temperature of 37°C for 8-10 h.

[0052] (2) Induction: After culturing on a shaking table for 8-10 h, 2 mL of the bacterial solution was taken out and its OD600 value was measured by a spectrophotometer. When the OD600 value of the bacterial solution reached 0.6-0.8, IPTG was added to the bacterial solution at a final concentration of 200 μL / mL, and then the culture was shaken at 37°C and 170 rpm for 8 h.

[0053] (3) Purification: After adding IPTG and culturing on a shaking platform for 8 h, the bacterial solution was removed and centrifuged at 8000 rpm for 5 min at 4°C. After centrifugation, the supernatant was removed and the precipitate was retained (the precipitate is Escherichia coli).

[0054] (4) Ultrasonic disruption: The E. coli was ultrasonically disrupted and then centrifuged. The precipitate obtained by centrifugation contained the target protein. The precipitate obtained by centrifugation was first washed with washing solution I (50 mmol / L Tris-HCl, 1 mol / L urea, 10 mL / L Triton X-100), then washed with washing solution II (50 mmol / L Tris-HCl, 2 mol / L urea, 5 mL / L Triton X-100), and then dissolved with inclusion body solubilization solution (50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L NaCl).

[0055] (5) Finally, the inclusion body solution is subjected to gradient renaturation as follows: the inclusion body solution is placed in a dialysis bag, and the dialysis bag is placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in turn for gradient renaturation. The renaturation takes 2-4 hours at each urea concentration, and the renaturation must be performed at a low temperature of 4°C. The renaturation solution is purified by His-tag protein purification resin (nickel column) to obtain the new recombinant protein TAT-FLG2 (a histidine tag is added to the gene sequence of the target protein during design to facilitate protein purification). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify whether the purified target protein is successfully obtained. The results are as follows. Figure 2 As shown, a recombinant protein TAT-FLG2 with a size of about 28 kDa was obtained, which is consistent with its theoretical relative molecular mass.

[0056] The hydrophilicity analysis of the prepared new recombinant protein TAT-FLG2 was carried out using online analysis software (https: / / www.detaibio.com / tools / hydropathy-analysis.html). The results are shown in Figure 3 , the analysis data are as follows.

[0057] Table 2 Information on the novel recombinant protein TAT-FLG2 Experimental results show that the new recombinant protein TAT-FLG2 has good hydrophilicity and can be well dissolved in water. It can be subsequently prepared into various dosage forms such as aqueous solutions, emulsions, gels, and sprays.

[0058] Example 2 In this example, the cytotoxicity of the novel recombinant protein TAT-FLG2 was evaluated.

[0059] The toxicity of the new recombinant protein TAT-FLG2 on human embryonic kidney cells 293T and mouse fibroblasts L929 was detected by CCK8 method to evaluate the biosafety of the recombinant protein TAT-FLG2.

[0060] In a 96-well plate, 1 × 10 4Human embryonic kidney cells 293T (mouse fibroblast L929 experimental steps are the same as 293T cell experimental steps) were added to DMEM culture medium, and 5 experimental groups and 1 control group were set up, with 3 replicates in each group. After culturing the cells at 37°C for 24 hours, the new recombinant protein TAT-FLG2 was added to the wells of the experimental group (experimental wells), so that the final concentrations of the new recombinant protein TAT-FLG2 in the 5 experimental groups were 10μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, and 400μg / mL, respectively. No recombinant protein TAT-FLG2 was added to the wells of the control group (control wells). The cells were cultured at 37°C for 24 hours, and then the number of live cells was detected using the CCK8 method. The operation was to add 10μL of CCK8 reagent to each well and incubate at 37°C for 3 hours. The absorbance of each well at 450nm was then measured using an ELISA reader.

[0061] The cell viability was calculated as follows: [absorbance of the experimental well (culture medium containing cells, CCK8 reagent, protein sample)-absorbance of the blank well (culture medium without cells and protein sample, CCK8 reagent)] / [absorbance of the control well (culture medium containing cells, CCK8 reagent, no protein sample)-absorbance of the blank well (culture medium without cells and protein sample, CCK8 reagent)]×100%.

[0062] Results Figure 4 , Figure 4 The cell survival rate of each group is the average of the cell survival rates of the three replicate wells in the group. The cell survival rate of the control group was taken as 100%, where the horizontal axis was the concentration of the new recombinant protein TAT-FLG2 in the culture medium (μg / mL), and the vertical axis was the cell survival rate (%). The results showed that 293T cells and L929 cells showed a high survival rate after adding the recombinant protein TAT-FLG2 at a concentration of 10-400μg / mL. When the added concentration reached 400μg / mL, the cell survival rate was not less than 80%. It proves that the new recombinant protein TAT-FLG2 has low cytotoxicity and good biocompatibility.

[0063] Example 3 In this example, the cell penetration ability of the novel recombinant protein TAT-FLG2 was detected.

[0064] The experimental steps are as follows: (1) FITC labeling: 200 μg / mL of the novel recombinant protein TAT-FLG2 and natural filaggrin 2 were saturatedly labeled with FITC.

[0065] (2) Removal of free FITC: The FITC-labeled new recombinant protein TAT-FLG2 and natural filaggrin 2 were placed in dialysis bags MD25 (8000-14000D) respectively, and then the dialysis bags were dialyzed in a neutral PBS solution at 4°C for a total of 3 times, each time for 4 hours, to remove free FITC.

[0066] (3) Co-culture with HUVEC cells: The novel recombinant protein TAT-FLG2 and natural filaggrin 2 labeled with FITC were collected by ultracentrifugation (centrifugal force 16000g), and the collected novel recombinant protein TAT-FLG2 and natural filaggrin 2 were dissolved in complete culture medium at a concentration of 200 μg / mL. The complete culture medium solutions containing 200 μg / mL novel recombinant protein TAT-FLG2 and natural filaggrin 2 were used to co-culture HUVEC cells for 4 hours.

[0067] (4) Measurement of the average fluorescence density of HUVEC cells: After 4 hours of co-culture, HUVEC cells in the novel recombinant protein TAT-FLG2 experimental group and the natural filaggrin 2 experimental group were collected by centrifugation, and the average fluorescence intensity of the cells in the two experimental groups was measured using a fluorescence spectrophotometer.

[0068] The experimental results are shown in Figure 5 The results showed that the average fluorescence intensity of HUVEC cells in the experimental group of the new recombinant protein TAT-FLG2 was significantly higher than that in the experimental group of the natural filaggrin 2, further confirming that compared with the natural filaggrin 2, the new recombinant protein TAT-FLG2 has stronger cell penetration ability.

[0069] Example 4 This example provides a cell scratch test of the novel recombinant protein TAT-FLG2.

[0070] The experimental steps are as follows: (1) After L929 cells are revived, they are plated in a 24-well plate. When the L929 cells have grown to about 80%, use a yellow pipette tip (200 μL yellow tip) to vertically scratch the cells.

[0071] (2) 200 μg / mL of the novel recombinant protein TAT-FLG2 and natural filaggrin 2 were added to the scratched wells and co-cultured with L929 cells. Five parallel experiments were set up for each of the novel recombinant protein TAT-FLG2 and natural filaggrin 2 experimental groups.

[0072] (3) The healing rates of the scratches in the parallel experimental wells of each experimental group were measured at 6 h, 12 h, 18 h and 24 h using an ordinary optical microscope. The final healing rate of the scratches in each experimental group was based on the average healing rate of the five experimental wells.

[0073] The experimental results are shown in Figure 6 After about 24 hours of the experiment, the healing rate of cell scratches in the experimental group with the new recombinant protein TAT-FLG2 was close to 100%, while the healing rate of cell scratches in the experimental group with natural filaggrin 2 was less than 75%. The results showed that compared with natural filaggrin 2, the new recombinant protein TAT-FLG2 can more efficiently promote the healing of L929 cell scratches, which means that the new recombinant protein TAT-FLG2 also has the effect of promoting wound healing.

[0074] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0075] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A novel recombinant protein TAT-FLG2, characterized in that: The novel recombinant protein TAT-FLG2 comprises a partial peptide segment of filaggrin 2 and a cell-penetrating peptide; the amino acid sequence of the novel recombinant protein TAT-FLG2 is shown in SEQ ID NO.

1.

2. The novel recombinant protein TAT-FLG2 according to claim 1, characterized in that: The partial peptide segment of filaggrin 2 and the cell-penetrating peptide are connected via a flexible linker.

3. The novel recombinant protein TAT-FLG2 according to claim 1, characterized in that: The three-dimensional structure of the novel recombinant protein TAT-FLG2 has a V-like shape.

4. A recombinant expression vector, characterized in that: A gene comprising the novel recombinant protein TAT-FLG2 according to claim 1.

5. A recombinant bacterium, characterized in that Expressing the novel recombinant protein TAT-FLG2 according to any one of claims 1 to 3.

6. Use of the novel recombinant protein TAT-FLG2 according to any one of claims 1 to 3 in the preparation of external skin preparations.

7. The use according to claim 6, characterized in that The skin external preparation includes a spray, an aqueous solution, a gel or an emulsion.

8. A spray for promoting wound healing, characterized in that: The spray contains the novel recombinant protein TAT-FLG2 according to any one of claims 1 to 3.

9. The spray according to claim 8, characterized in that The spray may also contain other pharmaceutically acceptable adjuvants and / or auxiliary agents.

10. A spray for promoting fibroblast migration, characterized in that: The spray contains the novel recombinant protein TAT-FLG2 according to any one of claims 1 to 3.

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