A recombinant humanized filaggrin with good moisturizing effect, its preparation method and application

Abstract

The present invention belongs to the field of bioengineering, and particularly relates to a recombinant humanized filaggrin with good moisturizing effect, a preparation method thereof, and an application thereof. By screening different fragments of natural filaggrin and expressing recombinant humanized filaggrin FLG using Escherichia coli, the present invention successfully prepares a recombinant humanized filaggrin FLG with good moisturizing effect, and its amino acid sequence is selected from one of SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15. It has been proven by experiments that the recombinant humanized filaggrin prepared by the present invention not only has good cell adhesion and cell migration activities, but also has good actual moisturizing experimental effects when applied to the human body, and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a recombinant humanized filaggrin with good moisturizing effect, a preparation method thereof and an application thereof. Background Art

[0002] Human filaggrin-2 (also known as ifapsoriasin) is a member of the S100 fusion protein (SFTP) family. The filaggrin-2 gene is encoded within the epidermal differentiation complex (EDC) on human chromosome 1q21.3. The gene consists of three exons, where the first small exon (about 50 bp in humans) is non-coding. The second exon (about 150-160 bp in humans) carries the start codon and the coding sequence of the S100 domain, while the very large third exon (up to several kb) carries the EF hand domain and the remaining coding sequence.

[0003] The filaggrin-2 protein sequence is up to 2391 amino acids long, with a corresponding molecular mass of 248 kDa and a pI of 8.45. The N-terminal domain of filaggrin-2 is homologous to the N-terminal domain of S100A proteins, followed by a large repetitive region containing two types of tandem repeats, 9 a-type repeats and 14 b-type repeats. These two types are similar in length (75 or 77 amino acids) but different in sequence. They are rich in serine (about 32%) and glycine (about 28% and 17%).

[0004] Different regions of the filaggrin-2 structure have different biological activities. The N-terminal domain of filaggrin-2 can bind to Ca 2+ ions. The 9 a-type repeats (A1-A9) are homologous to keratin repeats (50-77% homology), and keratin is a confirmed component of the cell envelope. The 14 b-type repeats (B1-B14) are more similar to the FLG unit (28-39% homology), rich in glutamine (8.4%), basic amino acids Arg (8.4%) and His (14.9%). Although the b-type repeats are not rich in lysine, they contain a large amount of glutamine (about 15% and 8% respectively). These amino acids are candidate amino acids for the glutamine receptor sites that form protein-protein crosslinks during keratinocyte differentiation. In addition, FLG2 has been shown to accumulate in keratin granules like FLG and HRNR and undergoes proteolytic processing during later keratinization.

[0005] Current research has found that filaggrin-2 is not only expressed as a structural component during keratinocyte differentiation, but filaggrin-2 has also been identified to interact with a variety of different molecules, such as Rho kinase 1 (ROCK1), epidermal growth factor receptor (EGFR) antagonists, human cytomegalovirus DNA polymerase subunit UL44, proliferating cell nuclear antigen (PCNA)-associated factor (PAF15), cyclin D1, and integrin β1 complex. As an interactor and regulator of various cell processes related to differentiation, for example, it affects cell cycle regulation and proliferation in a certain way. It maintains the integrity of the skin barrier and is also a susceptibility factor for certain diseases, or can compensate for those diseases characterized by limited or lost filaggrin function.

[0006] In addition, some studies have shown that filaggrin-2 plays an important role in anti-skin aging. These studies have shown that a decrease in filaggrin-2 levels is associated with skin aging and the occurrence of skin lesions.

[0007] The epidermis, as the outermost barrier of the skin, is in long-term contact with external factors. For the barrier function of the skin, it is important to ensure the integrity of the barrier by maintaining or improving the differentiation process of keratinocytes and to respond immediately to environmental and internal challenges (such as xenobiotics or toxic substances). In the upper layer of the stratum corneum, citrullinated filaggrin-2 monomers are degraded into individual amino acids, which are then further processed into uric acid (UCA, derived from histidine) and pyrrolidone carboxylic acid (PCA, derived from glutamine), serving as hygroscopic "natural moisturizing factors" of the skin to retain moisture and protect against ultraviolet rays.

[0008] Therefore, by increasing the level of filaggrin-2, the health of the skin can be improved, which plays an important role in maintaining the skin barrier function, keeping the skin moist, and skin health. Summary of the Invention

[0009] Based on this, the present invention provides a recombinant humanized filaggrin with good moisturizing effect. The present invention realizes the soluble expression of recombinant human filaggrin, reduces the expression cost, effectively shortens the expression cycle, improves the product performance, and expands its application scope.

[0010] In order to achieve the above technical effects, the technical solution adopted by the present invention is as follows:

[0011] A recombinant humanized filaggrin with good moisturizing effect, wherein the amino acid sequence of the recombinant humanized filaggrin is selected from one of SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15.

[0012] The present invention also provides a polynucleotide sequence encoding the recombinant humanized filaggrin, and the polynucleotide sequence is selected from one of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0013] In the present invention, the original nucleotide sequences encoding the recombinant humanized filaggrin are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively. Then the codons are optimized, and six histidine codons are added to the 3' end. The finally optimized sequences are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0014] The present invention also provides a recombinant humanized filaggrin expression vector, which includes the polynucleotide sequence encoding the recombinant humanized filaggrin.

[0015] The present invention also provides a recombinant engineering bacterium, which includes the recombinant humanized filaggrin expression vector.

[0016] Preferably, the host of the recombinant engineering bacterium is Escherichia coli.

[0017] The present invention also provides a preparation method of the recombinant humanized filaggrin, which includes the following processes:

[0018] S1. Synthesize the polynucleotide sequences of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 by biosynthesis method to obtain DNA target fragments;

[0019] S2. Double digest the DNA target fragments obtained in step S1 with XbaI and NcoI, and then insert them into the shuttle vector PET28a(+) to construct recombinant plasmids PET28a(+)-FLG-1, PET28a(+)-FLG-2, PET28a(+)-FLG-3, PET28a(+)-FLG-4, and PET28a(+)-FLG-5;

[0020] S3. Transfer the recombinant plasmids PET28a(+)-FLG-1 to PET28a(+)-FLG-5 obtained in S2 into Escherichia coli respectively to obtain positive bacteria, and further culture and induce expression to obtain the bacterial bodies containing the recombinant humanized filaggrin FLG;

[0021] S4, Purification: Resuspend and disrupt the bacterial cells obtained in step S3; centrifuge to collect the supernatant, filter it, and then purify the filtered solution by affinity chromatography using a nickel column to obtain the product.

[0022] Preferably, the process of affinity chromatography purification in step S4 is as follows: Pass the filtrate containing recombinant humanized filaggrin FLG through a nickel column, wash away the unbound impurities with the binding buffer and the impurity washing buffer, and elute the recombinant humanized filaggrin FLG with the elution buffer; add it to a G25 desalting column and wash it out with PBS buffer to obtain highly pure recombinant humanized filaggrin FLG.

[0023] Preferably, the components of the binding buffer include 0.4 - 0.6 M NaCl, 45 - 55 mM PB, 15 - 25 mM imidazole, 7 - 9 M urea, pH = 8.3; the components of the impurity washing buffer include 0.4 - 0.6 M NaCl, 45 - 55 mM PB, 70 - 90 mM imidazole, 3 - 5 M urea, pH = 8.3; the components of the elution buffer include 0.4 - 0.6 M NaCl, 45 - 55 mM PB, 250 - 350 mM imidazole, 2 M urea, pH = 8.3; the components of the desalting buffer include 0.1 - 0.2 M NaCl, 45 - 55 mM PB, pH = 6.5.

[0024] The present invention also provides an application of the recombinant humanized filaggrin as described above in the preparation of a biological skin moisturizing preparation.

[0025] Preferably, the biological skin moisturizing preparation is one of freeze-dried powder, biological sponge or dressing.

[0026] Based on the sequence structure and functional information of filaggrin-2, the present invention selects the sequence part rich in histidine and glutamine as the research object. Analyze the amino acid sequence of the full-length filaggrin by means of bioinformatics and other methods, and design a series of new filaggrin sequences containing 961, 480, 371, 277, and 195 amino acids respectively through long-term experimental screening. The sequences cover multiple active sites and can better exert the biological function of human filaggrin.

[0027] The epidermis, as the outermost barrier of the skin, is in long-term contact with external factors. For the skin's barrier function, it is important to ensure barrier integrity by maintaining or improving the differentiation process of keratinocytes and to respond immediately to environmental and internal challenges (such as xenobiotics or toxic substances). In the upper layer of the stratum corneum, citrullinated filaggrin-2 monomers are degraded into individual amino acids, which are then further processed into uric acid (UCA, derived from histidine) and pyrrolidone carboxylic acid (PCA, derived from glutamine), acting as hygroscopic "natural moisturizing factors" of the skin to retain moisture, protect against ultraviolet rays, and prevent skin aging. Therefore, by increasing the level of filaggrin-2, the health of the skin can be improved, playing an important role in maintaining skin barrier function and skin health.

[0028] Escherichia coli is a Gram-negative bacterium that can grow in a simple culture medium and usually reproduces once every 20 minutes, making its culture operation relatively simple, with low cost and a short fermentation cycle. Additionally, since its genetic background has been widely studied and its genomic sequence has been fully sequenced, it is very convenient to manipulate its genetic material at the molecular level, and it is one of the most commonly used host cells in biotechnology. Moreover, the protein expression system of Escherichia coli has a high transfection efficiency, can produce a large amount of recombinant protein, and the level of the expressed foreign gene product is much higher than that of other gene expression systems, even reaching more than 30% of the total bacterial protein. This makes it widely used in many fields such as medicine, biotechnology, and agriculture, including the production of prophylactic vaccines, therapeutic recombinant proteins, therapeutic enzymes, medical aesthetic raw materials, scientific research enzymes, and industrial enzymes, and it is an ideal host for large-scale production of recombinant proteins. Therefore, the present invention selects Escherichia coli as the host bacterium for the expression of recombinant humanized filaggrin.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The recombinant humanized filaggrin obtained in the present invention not only has high biological activity, but also can achieve higher expression efficiency and higher stability;

[0031] (2) The nucleotide sequence of the recombinant filaggrin of the present invention is optimized according to the codon preference of Escherichia coli, and a high-expression recombinant strain is constructed. The recombinant filaggrin produced by the fermentation of this recombinant strain has exactly the same protein sequence as natural filaggrin and is a recombinant humanized filaggrin with good safety;

[0032] (3) The present invention provides a low-cost and highly efficient protein purification process, which is suitable for large-scale production;

[0033] (4)The recombinant humanized filaggrin prepared by the present invention has good skin moisturizing ability and can be well applied to the cosmetic field. At the same time, the recombinant humanized filaggrin provided by the present invention can also be used for the research of skin health and diseases, and has broad application prospects. Brief Description of the Drawings

[0034] Figure 1 It is the gel electrophoresis band pattern of the selected positive clone bacteria PET28a(+)-FLG-1 to PET28a(+)-FLG-3 before and after induction.

[0035] Figure 2 It is the gel electrophoresis band pattern of the selected positive clone bacteria PET28a(+)-FLG-4 and PET28a(+)-FLG-5 before and after induction.

[0036] Figure 3 It is the SDS-PAGE pattern of each purified recombinant filaggrin FLG-1, FLG-2, FLG-3, FLG-4, FLG-5.

[0037] Figure 4 It is the result diagram of the influence of the recombinant filaggrin FLG-1, FLG-2, FLG-3, FLG-4, FLG-5 of the present invention on cell adhesion.

[0038] Figure 5 It is the result diagram of the influence of the recombinant filaggrin FLG-1, FLG-2, FLG-3, FLG-4, FLG-5 of the present invention on cell migration.

[0039] Figure 6 It is a schematic diagram of the measurement parts for the moisturizing effect test.

[0040] Figure 7 It is the descriptive statistical result diagram of the water content of the stratum corneum of the flexor skin of the forearm.

[0041] Figure 8 It is the result diagram of the difference analysis of the water content of the stratum corneum of the flexor skin of the forearm.

[0042] Figure 9 It is the result diagram of the change in the water content of the stratum corneum of the flexor skin of the forearm before and after use. Detailed Embodiments

[0043] The present invention will be further explained below in conjunction with specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions identical or similar to the present invention are within the protection scope of the present invention. For those not specifying specific technologies or conditions in this embodiment, operations shall be conducted according to the conventional technical methods and instrument specification contents in the art; for reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0044] The buffer solutions involved in the experiment are as follows:

[0045] The components of 50 mM PB (i.e., PB involved below) include 2.65 mM sodium dihydrogen phosphate dihydrate + 47.35 mM disodium hydrogen phosphate dodecahydrate, pH = 8.3; the components of the binding buffer (equilibration buffer) include 0.5 M NaCl, 50 mM PB, 20 mM imidazole, 8 M urea, pH = 8.3; the components of the washing buffer include 0.5 M NaCl, 50 mM PB, 80 mM imidazole, 4 M urea, pH = 8.3; the components of the elution buffer include 0.5 M NaCl, 50 mM PB, 300 mM imidazole, 2 M urea, pH = 8.3; the components of the desalting buffer include 0.15 M NaCl, 50 mM PB, pH = 6.5.

[0046] Example 1 Design of the recombinant humanized filaggrin sequence

[0047] The amino acid sequence of natural human filaggrin (Entry: Q5D862) was obtained using the uniprot database. The sequence was analyzed by bioinformatics methods to identify the amino acid enrichment regions in the original sequence, find the corresponding active sites in the sequence, and design the corresponding protein sequence based on the amino acid characteristics and the predicted spatial structure of the protein sequence. A total of 5 recombinant filaggrins were finally designed in the present invention, containing 961, 480, 371, 277, and 195 amino acids from large to small, respectively. The specific amino acid sequences are shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15. These sequences not only contain integrin binding sites and the content of active amino acids such as glutamine and histidine, ensuring the biological function of the recombinant filaggrin, but also have good water solubility and stability, enabling efficient expression. The sequences all contain histidine tags, facilitating subsequent purification.

[0048] Example 2 Construction of the recombinant bacterium E. coli BL21 / PET28a(+)-FLG

[0049] According to the codon preference of Escherichia coli, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 in Example 1 (the sequence information is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5) were codon-optimized. The optimized nucleotide sequences of the encoding genes are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, named as the nucleotide sequences of FLG-1, FLG-2, FLG-3, FLG-4, FLG-5. They were entrusted to Sangon Biotech for synthesis. Then the synthesized target genes were cloned onto the Escherichia coli expression vector pET28a(+), and then transformed into competent E. coli (DH5α). Positive clones were obtained by screening, and then plasmids were extracted to obtain the recombinant expression vectors pET28a(+)-FLG-1, pET28a(+)-FLG-2, pET28a(+)-FLG-3, pET28a(+)-FLG-4, pET28a(+)-FLG-5. After DNA sequencing and alignment, the recombinant expression vectors pET28a(+)-FLG-1, pET28a(+)-FLG-2, pET28a(+)-FLG-3, pET28a(+)-FLG-4 and pET28a(+)-FLG-5 were successfully constructed.

[0050] Subsequently, the successfully sequenced plasmids pET28a(+)-FLG-1, pET28a(+)-FLG-2, pET28a(+)-FLG-3, pET28a(+)-FLG-4, pET28a(+)-FLG-5 were respectively transferred into the Escherichia coli expression strain BL21(DE3). Through screening on kanamycin-resistant LB plates, the recombinant strains E. coli BL21 / pET28a(+)-FLG-1, E. coli BL21 / pET28a(+)-FLG-2, E. coli BL21 / pET28a(+)-FLG-3, E. coli BL21 / pET28a(+)-FLG-4, E. coli BL21 / pET28a(+)-FLG-5 were obtained.

[0051] Example 3 Induced expression of the recombinant strain E. coli BL21 / pET28a(+)-FLG

[0052] Select single colonies of the recombinant bacteria E. coli BL21 / PET28a(+)-FLG-1, E. coli BL21 / PET28a(+)-FLG-2, E. coli BL21 / PET28a(+)-FLG-3, E. coli BL21 / PET28a(+)-FLG-4, and E. coli BL21 / PET28a(+)-FLG-5 screened in Example 2 and inoculate them into LB liquid medium (containing 50 μg / mL kanamycin) for cultivation. The cultivation conditions are 37°C and 250 rpm. When the cell density reaches OD600 = 0.6, take a sample (the sampling amount is half of the total cells), centrifuge to collect the cells, and label it as the sample before induction. Then add IPTG with a final concentration of 0.5 mmol to the remaining bacterial solution for induction. The induction conditions are 25°C and 250 rpm for 4 h. Then centrifuge at 12,000 rpm for 5 min to collect the cells, and label it as the sample after induction. The samples before and after the above induction are resuspended with lysozyme solution, the cells are lysed by digestion at 37°C for 10 min, and then SDS-PAGE detection is carried out. The theoretical sizes of the screened recombinant humanized filaggrin are 101.52 kDa, 49.67 kDa, 39.60 kDa, 29.58 kDa, and 20.75 kDa, respectively.

[0053] The results are as Figure 1 and Figure 2 shown, Figure 1 in which, band 1 is the detection result before induction of PET28a(+)-FLG-1, band 2 is the detection result after induction of PET28a(+)-FLG-1, band 3 is the result before induction of PET28a(+)-FLG-2, band 4 is the result after induction of PET28a(+)-FLG-2, band 5 is the result before induction of PET28a(+)-FLG-3, and band 6 is the result after induction of PET28a(+)-FLG-3. Figure 2 In

[0054] which, band 7 is the result before induction of PET28a(+)-FLG-4, band 8 is the result after induction of PET28a(+)-FLG-4, band 9 is the result before induction of PET28a(+)-FLG-5, and band 10 is the result after induction of PET28a(+)-FLG-5.

[0055] Example 4 Purification of Recombinant Humanized Filaggrin

[0056] The positive clone recombinant bacteria E. coli BL21 / PET28a(+)-FLG-1, E. coli BL21 / PET28a(+)-FLG-2, E. coli BL21 / PET28a(+)-FLG-3, E. coli BL21 / PET28a(+)-FLG-4, and E. coli BL21 / PET28a(+)-FLG-5 screened in Example 2 were fermented in 250 mL shake flasks for 16 h. The obtained fermentation broth was centrifuged at 10,000 rpm for 20 min using a high-speed centrifuge to obtain cell pellets. The cell pellets were purified through the following steps to obtain pure recombinant humanized filaggrin:

[0057] ① Cell disruption: The cell pellets were resuspended in a solution with a mass-volume ratio of 1:6 (50 mM PB + 500 mM NaCl, pH = 8.3), and then the cells were disrupted using a homogenizer at a pressure of 800 bar for 3 times to obtain a cell lysate containing the target protein.

[0058] ② Collection of cell lysate: The cell lysate containing the target protein after the above disruption was centrifuged at 4 °C and 20,000 rpm for 20 min. The precipitate was removed, and the supernatant was collected, which was the cell lysate containing the target protein.

[0059] ③ Nickel column purification: The cell lysate containing the target protein collected in step ② above was purified using a nickel column. The purification method was as follows:

[0060] 1) Nickel column equilibration: First, the column was rinsed with 3 column volumes (CV) of deionized water to remove the protective solution (20% ethanol) in the column, and then the column was rinsed with 3 CV of equilibration buffer (i.e., binding buffer).

[0061] 2) Loading of cell lysate: The cell lysate containing the target protein collected in ② was filtered through a 0.45 μm membrane and then loaded onto the column. The loading volume was 2 CV.

[0062] 3) Removal of impurity proteins: The column was rinsed with washing buffer to remove impurity proteins until the UV280 curve became flat.

[0063] 4) Elution of target protein: The target protein was eluted using elution buffer. According to the appearance of a new peak in the UV280, collection was started until the collection peak became flat.

[0064] 5) SDS-PAGE detection: The elution sample of the target protein above was subjected to SDS-PAGE detection. After passing the detection (the size of the protein band was as expected), the next step was carried out.

[0065] ④Molecular sieve purification (G25 packing): Further purify the target protein that has passed the detection in nickel column purification 5). The purification method is as follows:

[0066] 1) Molecular sieve column equilibration: First, rinse the column with 3 CV of deionized water to remove the protective solution (20% ethanol) in the column, and then rinse the column with desalting buffer for 3 CV;

[0067] 2) Sample loading: Load the target protein that has passed the above detection, and the sample loading volume is 0.2 CV;

[0068] 3) Desalting: Continue to use the desalting buffer to desalt and rinse the column until the protein flows out, and collect the protein.

[0069] The test results are as Figure 3 shown, Figure 3 in which, band 1 is the purified product of FLG-1, band 2 is the purified product of FLG-2, band 3 is the purified product of FLG-3, band 4 is the purified product of FLG-4, and band 5 is the purified product of FLG-5. The results show that the purity of the protein purified by the above steps is basically greater than 90%.

[0070] Example 5 Cell Adhesion Test of Recombinant Filaggrin

[0071] 1. Sample preparation:

[0072] (1) Preparation of positive control (animal collagen) solution: Prepare an animal collagen (taking bovine collagen as an example) with a concentration of 1 μg / mL as the positive control. Sterilize it through a 0.22 μm filter membrane and store it at -20 °C for later use. Use ultrapure water as the negative control during the test and make 2 replicates. All the above operations are carried out under sterile conditions.

[0073] (2) Preparation of sample solution: Dilute the purified product of filaggrin finally obtained in Example 4 with ultrapure water to 1 μg / mL and make 2 replicates. All the above operations are carried out under sterile conditions.

[0074] (3) Preparation of PBS solution: Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 3.63 g of disodium hydrogen phosphate dodecahydrate, and 0.24 g of potassium dihydrogen phosphate. Add 800 mL of deionized water and dissolve it fully. Make up the volume to 1000 mL with a volumetric flask, sterilize it, and store it sealed at 4 °C.

[0075] (4) Preparation of complete medium: Take 10 mL of fetal bovine serum and 1 mL of penicillin-streptomycin double antibody, and add DMEM basal medium to make up to 100 mL.

[0076] 2. Test procedure: The specific operation process is as follows:

[0077] (1)Add 100 μL of positive control, negative control (ultrapure water), and sample solution to a 96-well plate, seal it, and incubate overnight at 4°C. When conducting the experiment, discard the liquid, wash it once with PBS solution, and set it aside;

[0078] (2)Digest HaCaT cells in the logarithmic phase with trypsin, resuspend them with complete medium containing 10% FBS, and add 100 μL of the diluted cell suspension to the above 96-well plate to make the cell density 15,000 cells / well.

[0079] (3)After inoculation for 3 h (which can be adjusted according to the cell status), observe the cell adhesion status under a microscope.

[0080] (4)Add 10 μL of CCK8 to each well, mix well, continue to culture for 2 h, then take it out of the incubator, place it on a microplate reader, and detect the absorbance at a wavelength of 450 nm. Record the measurement results.

[0081] (5)Process the data with Excel software, and the results are expressed as the adhesion rate. The calculation formula is:

[0082]

[0083] 3. Test results: The specific test results are as Figure 4 shown. The results indicate that 1 μg / mL of recombinant human filaggrin has good cell adhesion-promoting activity.

[0084] Example 6 Recombinant human filaggrin cell scratch test

[0085] 1. Preparation of test sample solution: Dilute bovine collagen, recombinant human filaggrin samples FLG-1, FLG-2, FLG-3, FLG-4, FLG-5 to 10 μg / mL with serum-free DMEM medium (Gibco, USA), and make 2 replicates. All the above operations are carried out under sterile conditions.

[0086] 2. Operation procedure:

[0087] (1)First, on the back of a 12-well plate, use a marker pen to draw reference lines that cross the center of the wells horizontally, with at least 2 lines passing through each well;

[0088] (2)Digest NIH3T3 cells in the logarithmic phase with trypsin, resuspend them with DMEM medium containing 10% FBS (Gibco, USA), and inoculate the cells into a 12-well plate at a density of 150,000 cells / mL and 1 mL / well;

[0089] (3)On the second day, after observing that the cells had grown to 90%, dilute the sample to 1 μg / mL with serum-free DMEM medium (Gibco, USA). Then aspirate the medium in the original 12-well cell plate, leaving 200 μL of medium. Using a 200-μL pipette tip against a ruler, make a scratch as perpendicular as possible to the reference line on the back. The pipette tip should be vertical and not tilted. Wash the cells 2 times with PBS to remove the detached cells. After observing under the microscope that there are no floating cells, it means the washing is clean. Then add the prepared sample, 1 mL per well, and make marks. Use a microscope (Leica, Germany) to record the cell scratch data, which is recorded as 0 hour. After recording, place the cells back into the cell incubator (37 °C, 5% CO2, Thermo Fisher, USA) for culture. Take pictures at 24 hours and 48 hours at the selected positions (the different time points for each group should be at the same position). The shooting time can be adjusted according to the actual situation;

[0090] (4)Process the pictures with ImageJ software, and the results are expressed as the migration rate. The calculation formula for the migration rate is as follows:

[0091]

[0092] 3. Test results: The test results are as Figure 5 shown. The results indicate that the recombinant human filaggrin at 1 μg / mL has good cell migration-promoting activity.

[0093] Example 7 Application of Recombinant Humanized Filaggrin

[0094] Based on the above experimental results of cell adhesion and migration activities, filaggrin FLG-2 was selected for the following human efficacy experiments. To verify the moisturizing effect of humanized filaggrin on the skin, that is, the moisturizing effect of recombinant filaggrin FLG-2 on the skin, this example was verified through the following tests:

[0095] 1. Subject screening

[0096] Number of subjects: The minimum effective sample size is 24 cases.

[0097] Inclusion criteria:

[0098] (1)Aged 18 - 65 years old, healthy men or women;

[0099] (2)Measured by a skin moisture tester, the baseline value in the forearm test area is between 15 and 45 (Corneometer Unit, C.U);

[0100] (3)The skin of the subjects' forearms is intact, without any scars, injuries or other factors affecting the evaluation;

[0101] (4)Able to strictly follow the requirements and time arrangements of the research protocol and sign the informed consent form.

[0102] Exclusion criteria:

[0103] (1) Women who are currently pregnant, lactating, or planning to become pregnant in the next two months;

[0104] (2) Those with severe systemic diseases, immunodeficiency, or autoimmune diseases, and those who have received skin treatments, beauty treatments, and other tests that may affect the results at the test site;

[0105] (3) Those with active allergic diseases or highly sensitive constitutions;

[0106] (4) Those who have used hormonal drugs and immunosuppressants within the last month;

[0107] (5) Those who are currently or have participated in other clinical trials at the test site in the last three months.

[0108] Withdrawal criteria: Severe adverse reactions occurred during the trial and failure to comply with the test protocol (such as using other cosmetics, drugs, and other reasons for withdrawal during the test).

[0109] 2. Preparation before the test:

[0110] No products (cosmetics or topical drugs) can be used on the test site within 3 days before the test, and water cannot be contacted within 3 hours before the test. 3 hours before the test, the subject needs to clean the flexor side of both forearms, gently wipe them clean with a paper towel, and sit quietly in the human efficacy evaluation room (temperature: 21°C ± 1°C, humidity: 50% ± 10%) for 30 minutes. Water and beverages cannot be consumed during the test. The forearms should be exposed and placed in the test state, keeping relaxed. Before the test, the subject should be explained about the test and sign the informed consent form.

[0111] The test area is the flexor side of both forearms, which is divided into five measurement areas: A, B, C, D, and E. The area of each area is at least 3 cm × 3 cm, and the interval between each test area is at least 1 cm. The tester selects the test product area, negative control area (using desalted buffer as the negative control), and blank control area on the right and left forearms in advance according to the random table, and the measurement sites are as Figure 6 shown.

[0112] 3. Product usage method: The tester uses disposable latex finger cots to evenly coat the test product and the negative control product on the designated test area and negative control area at a dosage of (2.0 ± 0.1) mg / cm² once according to the area setting, and no product is used in the blank control area.

[0113] 4. Objective quantitative evaluation: After wiping the arm with dry paper towels for 30 minutes, use the Delfin MoistureMeter SC to measure and analyze the water content of the stratum corneum of the skin in all the tested areas on the flexor side of the forearm as the initial value (T0h), and then use the product in the selected areas. The subjects need to be visited and the same index tests should be carried out 2 hours (T2h) and 4 hours (T4h) after using the product. The tests for the same subject should be completed by the same instrument and the same tester. The measurement probe should be cleaned between two measurements. The test site should be kept consistent before and after between the two test time points.

[0114] 5. Statistical analysis: Statistically analyze the measured values of each test area, including the quantity, mean, standard deviation, etc.

[0115] Calculate the difference between the initial value and the measured values at other time points in each test area, and then use this difference to statistically analyze the differences between the product area and the negative control area at different time points.

[0116] If the test data is normally distributed, the paired t-test method is used for statistical analysis; if the test data is not normally distributed, the rank sum test method is used for statistical analysis. The statistical method uses a two-tailed test, and the test level α = 0.05. The statistical data is statistically analyzed using SPSS 20.0 software. When the results show significant differences, they are marked with *p < 0.05 and **p < 0.01 respectively. *p < 0.05 indicates a significant difference, and **p < 0.01 indicates a highly significant difference. If there are significant differences in the numerical values of each index in the test area, it means that the product has a moisturizing effect. When the results show no significant differences, they are marked with ns, indicating that the product does not have a moisturizing effect.

[0117] 6. Test results:

[0118] (1) Safety: No cases of skin adverse reactions occurred in this test, indicating that the test product has good safety.

[0119] (2) Moisturizing effect, the specific results are as Figures 7 - 9 shown, among which, Figure 7 is the descriptive statistics of the water content of the stratum corneum of the skin on the flexor side of the forearm (C.U., n = 32); Figure 8 is the differential analysis of the water content of the stratum corneum of the skin on the flexor side of the forearm (C.U., x ± s, n = 32); Figure 9 is the change in the water content of the stratum corneum of the skin on the flexor side of the forearm before and after use (C.U., x ± s, n = 32). Thus, it can be seen that:

[0120] 1) Test product (50 ppm filaggrin): Analysis of the water content of the skin stratum corneum showed that after the subjects used the test product (50 ppm filaggrin), the water content of the skin stratum corneum on the flexor side of the forearm did not increase significantly at each follow-up time point T2h and T4h (p > 0.05), and the difference in the water content of the skin stratum corneum 2 hours and 4 hours after using the test product (50 ppm filaggrin) was not significantly different from that of the negative control group (p > 0.05). The results showed that the test product (50 ppm filaggrin) did not have a moisturizing effect at 2h and 4h.

[0121] 2) Test product (100 ppm filaggrin): Analysis of the water content of the skin stratum corneum showed that after the subjects used the test product (100 ppm filaggrin), the water content of the skin stratum corneum on the flexor side of the forearm increased extremely significantly at each follow-up time point T2h and T4h (p < 0.01), and the difference in the water content of the skin stratum corneum 2h and 4h after using the test product (100 ppm filaggrin) was extremely significantly different from that of the negative control group (p < 0.01).

[0122] 3) Test product (2000 ppm filaggrin): Analysis of the water content of the skin stratum corneum showed that after the subjects used the test product (2000 ppm filaggrin), the water content of the skin stratum corneum on the flexor side of the forearm increased extremely significantly at each follow-up time point T2h and T4h (p < 0.01), and the difference in the water content of the skin stratum corneum 2 hours and 4 hours after using the test product (2000 ppm filaggrin) was extremely significantly different from that of the negative control group (p < 0.01).

[0123] The final results showed that the test products (100 ppm and 2000 ppm filaggrin) had an obvious moisturizing effect at 2 hours and 4 hours.

[0124] It should be noted that the above-described embodiments should be understood as illustrative and not limiting the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. For those skilled in the art, without departing from the essence and scope of the present invention, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention.

Claims

1. A recombinant humanized filaggrin with good moisturizing effect, characterized in that, The amino acid sequence of the recombinant humanized filaggrin is shown in SEQ ID NO.

12.

2. A polynucleotide encoding the recombinant humanized filaggrin as claimed in claim 1, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

7.

3. A recombinant humanized filaggrin expression vector, characterized in that, Comprising the polynucleotide according to claim 2.

4. A recombinant engineering bacterium, characterized in that, Comprising the recombinant humanized filaggrin expression vector according to claim 3.

5. The recombinant engineered bacterium according to claim 4, wherein Using Escherichia coli as the host.

6. A method for preparing the recombinant humanized filaggrin as described in claim 1, characterized in that, Comprising the following process: S1. Synthesize the polynucleotide sequence of SEQ ID NO.7 by biosynthetic method to obtain the DNA target fragment; S2. Double-digest the DNA target fragment obtained in step S1 with XbaI and NcoI and insert it into the shuttle vector PET28a to construct the recombinant plasmid PET28a-FLG-2; S3. Transfer the recombinant plasmid PET28a-FLG-2 obtained in S2 into Escherichia coli to obtain positive bacteria, and further culture and induce expression to obtain the bacterial cells containing the recombinant humanized filaggrin; S4. Purification: Resuspend and lyse the bacterial cells of the recombinant humanized filaggrin obtained in step S3; Centrifuge to collect the supernatant, filter, and then purify the filtered solution by affinity chromatography through a nickel column to obtain the product.

7. The preparation method according to claim 6, characterized in that, The process of affinity chromatography purification in step S4 is as follows: Pass the filtrate containing the recombinant humanized filaggrin through a nickel column, wash away the unbound impurities with the binding buffer and the washing buffer, and elute the recombinant humanized filaggrin with the elution buffer; Add it to a G25 desalting column and wash it out with the desalting buffer to obtain the recombinant humanized filaggrin with high purity.

8. The preparation method according to claim 7, characterized in that, The components of the binding buffer include 0.4 - 0.6 M NaCl, 45 - 55 mM PB, 15 - 25 mM imidazole, and 7 - 9 M urea, pH = 8.3; The components of the washing buffer include 0.4 - 0.6 M NaCl, 45 - 55 mM PB, 70 - 90 mM imidazole, and 3 - 5 M urea, pH = 8.3; The components of the elution buffer include 0.4 - 0.6 M NaCl, 45 - 55 mM PB, 250 - 350 mM imidazole, and 2 M urea, pH = 8.3; The components of the desalting buffer include 0.1 - 0.2 M NaCl and 45 - 55 mM PB, pH = 6.

5.

9. Use of a recombinant humanized filaggrin as claimed in claim 1 in the preparation of a biological skin moisturizing preparation.

10. The application according to claim 9, characterized in that, The biological skin moisturizing preparation is one of a freeze-dried powder, a biological sponge, or a dressing.

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