A novel recombinant protein TAT-FLG2 with cell membrane penetrating ability and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a novel recombinant protein TAT-FLG2 with cell membrane penetrating ability and its application. The novel recombinant protein TAT-FLG2 provided by the present invention comprises a partial peptide segment of filaggrin 2 and a cell-penetrating peptide, which are connected by a flexible linker. The three-dimensional structure of the novel recombinant protein TAT-FLG2 has a V-like shape. The novel recombinant protein TAT-FLG2 provided by the present invention has the ability to efficiently penetrate cell membranes and promote fibroblast migration.

Description

Technical Field

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

[0002] Filaggrin (FLG) is a key molecule that connects keratin fibers in the stratum corneum of human skin. FLG monomers facilitate the regular assembly of keratin fibers, forming a solid physical barrier in the outermost layer of the epidermis, thereby preventing water loss and the invasion of external allergens. Filaggrin 2 (FLG2), a key isoform of filaggrin, is synthesized in keratinocytes and distributed throughout various locations. It is gradually degraded by enzymes during keratinocyte migration. Enzymatically degraded FLG2 transforms into small molecules required by the stratum corneum, such as natural moisturizing factor (NMF), which effectively maintains a certain level of moisture in the skin and promotes the proliferation and metabolism of fibroblasts. Furthermore, the degradation products of FLG2 contain a high concentration of histidine, which, upon further metabolism, can generate pyrrolidone-5-carboxylic acid and trans-uridine, effectively maintaining the skin's slightly acidic environment and resisting microbial invasion. However, direct application of FLG2 has the problem of difficulty penetrating stratum corneum cells and, consequently, of effectively functioning in the dermis. In the prior art, there are strategies for applying other small molecules externally to promote the increase in 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 a skin care product containing oat β-glucan hydrolyzate for increasing 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 a skin topical preparation containing rose polysaccharide that can increase the content of filaggrin. Currently, there is little research on FLG2 or preparations containing FLG2 that have high penetration ability or efficiency when applied directly into the skin.

[0003] TAT (AYGRKKRRQRRR), derived from the transactivator of transcription of the human immunodeficiency virus HIV-1, is a cell-penetrating peptide with the ability to efficiently deliver various bioactive substances into cells. Currently, there are no reports of applying TAT to filaggrin to improve its membrane permeability.

[0004] In summary, it is necessary to propose a recombinant filaggrin with strong cell penetrating ability to alleviate or partially alleviate the shortcomings of the existing technology. 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 solutions.

[0006] In one aspect, the present invention provides a novel recombinant protein.

[0007] A 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.

[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 use 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 types of the vector include plasmid vector, phage vector, adenovirus vector or baculovirus vector.

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

[0016] In some optional embodiments, the bacterial species include Escherichia coli, Bacillus subtilis or Pichia pastoris strains.

[0017] The application of the above-mentioned novel recombinant protein TAT-FLG2 in the preparation of external 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 excipients and / or adjuvants.

[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:

[0026] The present invention provides a novel recombinant protein, TAT-FLG2, which has the ability to efficiently penetrate cell membranes. Compared to native filaggrin 2, the modified recombinant protein TAT-FLG2 has stronger cell penetration ability and can therefore be prepared as a formulation for direct external use. Furthermore, the novel recombinant protein TAT-FLG2 of the present invention also promotes fibroblast migration and wound healing. Clearly, the novel recombinant protein TAT-FLG2 provided by the present invention has promising market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are 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 inventive work.

[0028] Figure 1 This is the crystal structure of the novel recombinant protein TAT-FLG2 predicted by Alphafold 3;

[0029] Figure 2 This is an SDS-PAGE image of the novel recombinant protein TAT-FLG2 purified in one of the examples of the present invention;

[0030] Figure 3This is a diagram showing the hydrophilicity analysis results of the novel recombinant protein TAT-FLG2 in one of the embodiments of the present invention;

[0031] Figure 4 The present invention provides experimental results of one embodiment of the present invention using different concentrations of the novel recombinant protein TAT-FLG2 to determine the effects on the survival rates of human embryonic kidney cells (293T) and mouse epithelial-like fibroblasts (L929);

[0032] Figure 5 This is the statistical result of the mean fluorescence intensity after the novel recombinant protein TAT-FLG2 was co-cultured with vascular endothelial cells for a certain period of time in one embodiment of the present invention;

[0033] Figure 6 This is the experimental result of promoting the wound healing efficiency of L929 cells using the novel recombinant protein TAT-FLG2 and natural filaggrin 2 in one embodiment of the present invention. DETAILED DESCRIPTION

[0034] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 efforts are within the scope of protection of the present invention.

[0035] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0037] 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.

[0038] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges 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.

[0039] Definition of noun:

[0040] 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.

[0041] Main materials and reagents:

[0042] 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.

[0043] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0044] 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.

[0045] 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 Solaibao Technology Co., Ltd. DMEM medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd.

[0046] All reagents were used according to the instructions.

[0047] Culture medium:

[0048] 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.

[0049] Preparation: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950ml of double-distilled water. Adjust the pH to 7.0 with sodium hydroxide solution and make up to 1L with double-distilled water. If preparing a solid medium, add agar at 1.5g / 100ml. Autoclave at 121°C for 30 minutes.

[0050] Experimental reagents not otherwise specified 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; experimental methods not otherwise specified are all conventional methods in the art and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions from relevant reagent manufacturers.

[0051] Example 1

[0052] This example provides an example of TAT-FLG2 gene design and protein expression.

[0053] 1. Genetic Design

[0054] The designed gene contains a cell-penetrating peptide (YGRKKRRQRRR) and a partial amino acid sequence of filaggrin 2. The protein expressed by this gene has 264 amino acids, and its sequence is shown in SEQ ID NO. 1. The nucleotide sequence encoding this protein is shown in SEQ ID NO. 2, with a total length of 792 bp. Furthermore, NdeI restriction sites (CATATG) and XhoI restriction sites (CTCGAG) were added to the 5' and 3' ends of the gene, respectively. The nucleotide sequence of the gene with restriction sites is shown in SEQ ID NO. 3, with a total length of 801 bp. The sequences involved in this example are shown in Table 1.

[0055] Table 1 Sequence information

[0056]

[0057] 2. Vector Construction

[0058] The pET-22b(+) plasmid was used as an expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO. 1) were digested with restriction endonucleases NdeI and XhoI, respectively. The target gene was then ligated into the pET-22b(+) vector via ligation to produce a ligation product. It will be appreciated that other vectors, such as bacteriophage, adenovirus, or baculovirus vectors, may also be used.

[0059] 3. Conversion

[0060] (1) Take the competent E. coli BL21 (DE3) cells out of the -80°C freezer and place them on ice for 5 minutes.

[0061] (2) After the glycerol containing the BL21(DE3) competent cells has melted, add the competent cells to the ligation product, pipette and pump 3-4 times to mix thoroughly, and let stand on ice for 30 minutes.

[0062] (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.

[0063] (4) Add 800 μL of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0064] (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.

[0065] (6) Pick a single colony and inoculate it into liquid LB medium containing 100 μg / mL ampicillin. After incubation 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 bacterial transformation methods can also be used, such as Bacillus subtilis or Pichia pastoris strains.

[0066] 4. Protein Expression and Purification

[0067] (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 Escherichia coli (positive clone) containing the target gene plasmid into the LB medium at a volume of 200 μL / L. Then, 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.

[0068] (2) Induction: After culturing on a shaking platform for 8-10 h, 2 mL of the bacterial solution was taken out and its OD600 value was measured by 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 the culture was continued on a shaking platform at 37°C and 170 rpm for 8 h.

[0069] (3) Purification: After adding IPTG and culturing on a shaking platform for 8 h, the bacterial solution was removed and centrifuged at 8000 rpm at 4°C for 5 min. After centrifugation, the supernatant was removed and the precipitate was retained (the precipitate is Escherichia coli).

[0070] (4) Ultrasonic disruption: 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 dissolution solution (50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L NaCl).

[0071] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag, and the dialysis bag was placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in sequence for gradient renaturation. The renaturation was performed for 2-4 hours at each urea concentration, and the renaturation was performed at a low temperature of 4°C. After the renaturation, the solution was purified by His-tag protein purification resin (nickel column) to obtain the new recombinant protein TAT-FLG2 (a histidine tag was added to the gene sequence of the target protein during the design to facilitate protein purification). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the purified target protein was successfully obtained. The results are shown in FIG. Figure 2 As shown, a recombinant protein TAT-FLG2 with a size of approximately 28 kDa was obtained, which is consistent with its theoretical relative molecular mass.

[0072] 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.

[0073] Table 2 Information on the novel recombinant protein TAT-FLG2

[0074]

[0075] 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.

[0076] Example 2

[0077] In this example, the cytotoxicity of the novel recombinant protein TAT-FLG2 was evaluated.

[0078] The toxicity of the novel recombinant protein TAT-FLG2 on human embryonic kidney cells 293T and mouse fibroblasts L929 was detected by CCK8 assay to evaluate the biosafety of the recombinant protein TAT-FLG2.

[0079] In a 96-well plate, 1 × 10 4 Human embryonic kidney 293T cells (mouse fibroblast L929 cells follow the same experimental procedures as for 293T cells) were cultured in DMEM medium. Five experimental groups and one control group were set up, with triplicate wells in each group. After incubation at 37°C for 24 hours, the novel recombinant protein TAT-FLG2 was added to the experimental wells (experimental wells) to achieve final concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL for the five experimental groups, respectively. No recombinant protein TAT-FLG2 was added to the control wells (control wells). Culture was continued at 37°C for 24 hours. Viable cell counts were then determined using the CCK8 assay. 10 μL of CCK8 reagent was added to each well and incubated at 37°C for 3 hours. The absorbance of each well at 450 nm was then measured using a microplate reader.

[0080] The cell viability was calculated as follows: [absorbance of experimental wells (culture medium containing cells, CCK8 reagent, protein sample)-absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)] / [absorbance of control wells (culture medium containing cells, CCK8 reagent, no protein sample)-absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)] × 100%.

[0081] See the results Figure 4 , Figure 4 The cell viability rate for each group is the average of the cell viability rates of three replicate wells in that group. The cell viability of the control group is plotted as 100%, with the concentration of the novel recombinant protein TAT-FLG2 in the culture medium (μg / mL) on the horizontal axis and the cell viability (%) on the vertical axis. The results show that both 293T and L929 cells exhibited high viability after addition of recombinant TAT-FLG2 at concentrations ranging from 10 to 400 μg / mL. At a concentration of 400 μg / mL, the cell viability did not fall below 80%. This demonstrates that the novel recombinant protein TAT-FLG2 has low cytotoxicity and good biocompatibility.

[0082] Example 3

[0083] In this example, the cell penetration ability of the novel recombinant protein TAT-FLG2 was detected.

[0084] The experimental steps are as follows:

[0085] (1) FITC labeling: 200 μg / mL of the novel recombinant protein TAT-FLG2 and natural filaggrin 2 were saturated labeled with FITC.

[0086] (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), and then the dialysis bags were placed in a neutral PBS solution for dialysis at a temperature of 4°C for a total of 3 times, each for 4 hours, to remove free FITC.

[0087] (3) Co-culture with HUVEC cells: The FITC-labeled novel recombinant protein TAT-FLG2 and natural filaggrin 2 were collected by ultracentrifugation (16,000 g centrifugal force) and dissolved in complete culture medium at a concentration of 200 μg / mL. The complete culture medium solutions containing 200 μg / mL of the novel recombinant protein TAT-FLG2 and natural filaggrin 2 were used to co-culture HUVEC cells for 4 hours.

[0088] (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.

[0089] 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.

[0090] Example 4

[0091] This example provides a cell scratch test of the novel recombinant protein TAT-FLG2.

[0092] The experimental steps are as follows:

[0093] (1) After L929 cells are revived, they are plated in a 24-well plate. When the L929 cells grow to about 80%, use a yellow pipette tip (200 μL yellow tip) to scratch the cells vertically.

[0094] (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.

[0095] (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.

[0096] The experimental results are shown in Figure 6 After approximately 24 hours of the experiment, the wound healing rate of cells in the experimental group treated with the novel recombinant protein TAT-FLG2 was close to 100%, while the healing rate of cells in the experimental group treated with native filaggrin 2 was less than 75%. These results indicate that the novel recombinant protein TAT-FLG2 is more effective in promoting wound healing in L929 cells than native filaggrin 2, indicating that the novel recombinant protein TAT-FLG2 also has a wound healing effect.

[0097] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0098] 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 methods. The above-mentioned specific implementation methods are merely illustrative and not 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 protected by 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; the partial peptide segment of filaggrin 2 and the cell-penetrating peptide are connected by a flexible linker; the three-dimensional structure of the novel recombinant protein TAT-FLG2 has a V-like shape.

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

3. A recombinant bacterium, characterized in that Expressing the novel recombinant protein TAT-FLG2 according to claim 1.

4. A spray for promoting wound healing, characterized in that: The spray contains the novel recombinant protein TAT-FLG2 according to claim 1.

5. The spray according to claim 4, wherein The spray may also contain other pharmaceutically acceptable excipients and / or adjuvants.

6. A spray for promoting fibroblast migration, characterized in that: The spray contains the novel recombinant protein TAT-FLG2 according to claim 1.

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