Recombinant humanized silk protein with good moisturizing effect as well as preparation method and application of recombinant humanized silk protein

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a recombinant humanized silk protein with a good moisturizing effect as well as a preparation method and application of the recombinant humanized silk protein. According to the invention, different fragments of natural sericin are screened, escherichia coli is utilized to express recombinant humanized sericin FLG, the recombinant humanized sericin FLG with a good moisturizing effect is successfully prepared, and the amino acid sequence of the recombinant humanized sericin FLG 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. The invention further discloses a preparation method of the recombinant humanized sericin FLG. Experiments prove that the recombinant humanized silk protein prepared by the invention not only has good cell adhesion and cell migration activity, but also has a good actual moisturizing experiment effect when being applied to a human body, and has a good actual application value.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a recombinant humanized filaggrin with good moisturizing effect and a preparation method and 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, of which the first small exon (approximately 50 bp in humans) is non-coding. The second exon (approximately 150-160 bp in humans) carries the start codon and the coding sequence for the S100 domain, while the very large third exon (up to several kb in length) carries the EF-hand domain and the remaining coding sequence.

[0003] The protein sequence of filaggrin-2 is 2391 amino acids long, corresponding to a molecular mass of 248 kDa and a pI of 8.45. The N-terminal domain of filaggrin-2 is homologous to that of the S100A protein, followed by a large repeat region containing two types of tandem repeats, 9 a-type repeats and 14 b-type repeats, which 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+ In combination, the nine a-type repeats (A1-A9) are homologous to keratin repeats (50-77% homology), which are components of the cell envelope. The 14 b-type repeats (B1-B14) are closer to the FLG unit (28-39% homology) and are rich in glutamine (8.4%), the basic amino acids Arg (8.4%), and His (14.9%). Although b-type repeats are not rich in lysine, they contain a large amount of glutamine (approximately 15% and 8%, respectively). These amino acids are candidates for 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 to be proteolytically processed later in the cornification process.

[0005] Current studies have found that filaggrin-2 is not only expressed as a structural component in 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) antagonist, human cytomegalovirus DNA polymerase subunit UL44, proliferating cell nuclear antigen (PCNA)-related factor (PAF15), cyclin D1 and integrin β1 complex, etc. As an interactor and regulator of various cellular processes related to differentiation, for example, influencing cell cycle regulation and proliferation in some way. Maintaining the integrity of the skin barrier 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-aging of the skin. These studies have shown that the 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 permanent contact with external factors. It is important for the barrier function of the skin to respond immediately to environmental and internal challenges (such as xenobiotics or toxic substances) by maintaining or improving the differentiation process of keratinocytes to ensure the integrity of the barrier. In the upper layers of the stratum corneum, citrullinated filaggrin-2 monomers are degraded into single amino acids, which are then further processed into uric acid (UCA, derived from histidine) and pyrrolidone carboxylic acid (PCA, derived from glutamine), which act as the hygroscopic "natural moisturizing factor" of the skin to retain moisture and protect against UV rays.

[0008] 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, maintaining skin moisture, 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 product performance, and expands its application range.

[0010] In order to achieve the above technical effects, the technical solution adopted by the present invention is as follows: 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 and SEQ ID NO.15.

[0011] The present invention also provides a polynucleotide sequence encoding the recombinant humanized filaggrin, wherein 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.

[0012] The original nucleotide sequences encoding the recombinant humanized filaggrin in the present invention 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. The codons are then optimized, and 6 histidine codons are added to the 3' end. The final optimized sequences are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0013] The present invention also provides a recombinant humanized filaggrin expression vector, comprising the polynucleotide sequence encoding the recombinant humanized filaggrin.

[0014] The present invention also provides a recombinant engineering bacterium, comprising the recombinant humanized filaggrin expression vector.

[0015] Preferably, the recombinant engineered bacteria uses Escherichia coli as a host.

[0016] The present invention also provides a method for preparing recombinant humanized filaggrin, comprising the following steps: 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 to obtain the target DNA fragments; S2, the target DNA fragment obtained in step S1 was double-digested with XbaI and NcoI and inserted 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; S3, respectively transferring the recombinant plasmids PET28a(+)-FLG-1 to PET28a(+)-FLG-5 obtained in S2 into Escherichia coli to obtain positive bacteria, further culturing and inducing expression to obtain bacteria containing the recombinant humanized filaggrin FLG; S4, purification: resuspend and crush the recombinant humanized filaggrin FLG obtained in step S3; collect the supernatant by centrifugation, filter, and then purify the filtered solution by affinity chromatography through a nickel column to obtain the obtained product.

[0017] Preferably, the affinity chromatography purification process described in step S4 is as follows: the filtrate containing the recombinant humanized filaggrin FLG is passed through a nickel column, unbound impurities are washed away with a binding buffer and a washing buffer, and the recombinant humanized filaggrin FLG is eluted with an elution buffer; added to a G25 desalting column, and washed out with a PBS buffer to obtain a high-purity recombinant humanized filaggrin FLG.

[0018] Preferably, the components of the binding buffer include 0.4~0.6MNaCl, 45~55mM PB, 15~25mM imidazole, 7~9M urea, and pH=8.3; the components of the washing buffer include 0.4~0.6MNaCl, 45~55mM PB, 70~90mM imidazole, 3~5M urea, and pH=8.3; the components of the elution buffer include 0.4~0.6MNaCl, 45~55mM PB, 250~350mM imidazole, 2M urea, and pH=8.3; the components of the desalting buffer include 0.1~0.2MNaCl, 45~55mM PB, and pH=6.5.

[0019] The present invention also provides an application of the recombinant humanized filaggrin in the preparation of a biological skin moisturizing preparation.

[0020] Preferably, the biological skin moisturizing preparation is one of freeze-dried powder, biological sponge or dressing.

[0021] According to 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. The amino acid sequence of the full-length filaggrin is analyzed by means of bioinformatics, and combined with long-term experimental screening, a series of new filaggrin sequences containing 961, 480, 371, 277 and 195 amino acids are designed, respectively. The sequences cover multiple active sites and can better exert the biological function of human filaggrin.

[0022] The epidermis, as the outermost barrier of the skin, is in constant contact with external factors. It is important for the barrier function of the skin to respond immediately to environmental and internal challenges (such as xenobiotics or toxic substances) by maintaining or improving the differentiation process of keratinocytes to ensure the integrity of the barrier. In the upper layers of the stratum corneum, citrullinated filaggrin-2 monomers are degraded into single amino acids, which are then further processed into uric acid (UCA, derived from histidine) and pyrrolidone carboxylic acid (PCA, derived from glutamine), which act as the hygroscopic "natural moisturizing factor" of the skin to retain moisture and protect against UV rays, preventing skin aging. Therefore, by increasing filaggrin-2 levels, the health of the skin can be improved, playing an important role in maintaining the skin barrier function and skin health.

[0023] Escherichia coli is a Gram-negative bacillus that can grow in a simple culture medium and usually reproduce a generation every 20 minutes, which makes its culture operation relatively simple, low cost and short fermentation cycle. In addition, because its genetic background has been widely studied and the genome sequence has been completely sequenced, it is very convenient to operate 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 high transfection efficiency, can produce a large amount of recombinant protein, and the level of exogenous gene products expressed is much higher than other gene expression systems, and can even reach more than 30% of the total bacterial protein amount, which makes it widely used in many fields such as medicine, biotechnology, and agriculture, including the production of preventive vaccines, therapeutic recombinant proteins, therapeutic enzymes, medical and beauty raw materials, scientific research enzymes and industrial enzymes, etc., and is an ideal host for large-scale production of recombinant proteins. Therefore, the present invention selects Escherichia coli as the host bacteria for the expression of recombinant humanized filamentous protein.

[0024] Compared with the prior art, the present invention has the following advantages: (1) The recombinant humanized filaggrin obtained by the present invention not only has high biological activity, but also can achieve higher expression efficiency and higher stability; (2) The nucleotide sequence of the recombinant filaggrin of the present invention is optimized by the codon preference of Escherichia coli to construct a high-expression recombinant strain. The recombinant filaggrin produced by the recombinant strain is completely consistent with the protein sequence of the natural filaggrin, and is a recombinant humanized filaggrin with good safety. (3) The present invention provides a low-cost and efficient protein purification process suitable for large-scale production; (4) The recombinant humanized filaggrin prepared by the present invention has excellent skin moisturizing ability and can be well applied in the field of cosmetics. 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

[0025] Figure 1 This is the spectrum of the gel bands of the screened PET28a(+)-FLG-1~PET28a(+)-FLG-3 positive clone bacteria before and after induction; Figure 2 This is the spectrum of the gel bands of the screened PET28a(+)-FLG-4 and PET28a(+)-FLG-5 positive clones before and after induction; Figure 3 is the SDS-PAGE profile of the purified recombinant filaggrin proteins FLG-1, FLG-2, FLG-3, FLG-4, and FLG-5; Figure 4This is a result diagram showing the effects of the recombinant filaggrin proteins FLG-1, FLG-2, FLG-3, FLG-4 and FLG-5 described in the present invention on cell adhesion; Figure 5 This is a result diagram showing the effects of the recombinant filaggrin proteins FLG-1, FLG-2, FLG-3, FLG-4 and FLG-5 described in the present invention on cell migration; Figure 6 Schematic diagram of the measurement site for the moisturizing effect test; Figure 7 This is a descriptive statistical result diagram of the water content of the stratum corneum of the skin on the flexor side of the forearm; Figure 8 This is the result of the analysis of the water content difference of the stratum corneum of the skin on the flexor side of the forearm; Fig. 9 This is a graph showing the changes in stratum corneum water content before and after use on the flexor skin of the forearm. DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in this embodiment, the operation is carried out according to the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0027] The buffers involved in the experiment are as follows: The components of 50mM PB (i.e., PB mentioned below) include 2.65mM sodium dihydrogen phosphate dihydrate + 47.35mM sodium dihydrogen phosphate dodecahydrate, pH=8.3; the components of binding buffer (equilibrium buffer) include 0.5MNaCl, 50mM PB, 20mM imidazole, 8M urea, pH=8.3; the components of washing buffer include 0.5MNaCl, 50mM PB, 80mM imidazole, 4M urea, pH=8.3; the components of elution buffer include 0.5MNaCl, 50mM PB, 300mM imidazole, 2M urea, pH=8.3; the components of desalting buffer include 0.15MNaCl, 50mM PB, pH=6.5.

[0028] Example 1 Design of recombinant humanized filaggrin sequence The amino acid sequence of natural human filaggrin was obtained using the uniprot database (Entry: Q5D862), and the sequence was analyzed by bioinformatics methods to mine the amino acid enrichment region in the original sequence, find the corresponding active site in the sequence, and design the corresponding protein sequence according to the amino acid characteristics and the predicted protein sequence spatial structure. The present invention finally designed a total of 5 recombinant filaggrin proteins, which contain 961, 480, 371, 277 and 195 amino acids from large to small, respectively. The specific amino acid sequences are shown in 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, active amino acid contents such as glutamine and histidine, which ensure the biological function of recombinant filaggrin, but also have good water solubility and stability, can achieve efficient expression, and the sequences all contain histidine tags, and subsequent purification is convenient and simple.

[0029] Example 2 Construction of recombinant E. coli BL21 / PET28a(+)-FLG According to the preference of Escherichia coli codons, 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, and SEQ ID NO.15 in Example 1 (sequence information is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5) were codon optimized, and the optimized encoding gene nucleotide sequences are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15. As shown in .10, the nucleotide sequences named FLG-1, FLG-2, FLG-3, FLG-4, and FLG-5 were commissioned to be synthesized by Sangon Biotechnology. The synthesized target genes were then cloned into 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 recombinant expression vectors PET28a(+)-FLG-1, PET28a(+)-FLG-2, PET28a(+)-FLG-3, PET28a(+) -FLG-4, and PET28a(+)-FLG-5. After DNA sequencing and comparison, the recombinant expression vectors PET28a(+)-FLG-1, PET28a(+)-FLG-2, PET28a(+)-FLG-3, PET28a(+)-FLG-4, and PET28a(+)-FLG-5 were successfully constructed.

[0030] Then, the successfully sequenced plasmids PET28a(+)-FLG-1, PET28a(+)-FLG-2, PET28a(+) -FLG-3, PET28a(+)-FLG-4, and PET28a(+)-FLG-5 were respectively transformed into the Escherichia coli expression bacteria BL21(DE3), and screened on kanamycin resistance LB plates to obtain recombinant bacteria E. coliBL21 / PET28a(+)-FLG-1, E. coliBL21 / PET28a(+)-FLG-2, E. coliBL21 / PET28a(+) -FLG-3, E. coliBL21 / PET28a(+)-FLG-4, and E. coliBL21 / PET28a(+)-FLG-5.

[0031] Example 3 Inducible expression of recombinant E. coli BL21 / PET28a(+)-FLG 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 obtained by screening in Example 2 were selected and cultured in LB liquid culture medium (containing 50 μg / mL kanamycin). The culture conditions were 37 ° C, 250 rpm. When the bacterial density reached OD600 = 0.6, samples (the sampling amount was half of the total bacterial cells) were collected by centrifugation and marked as pre-induction samples. Then, IPTG with a final concentration of 0.5 mmoL was added to the remaining bacterial solution for induction. The induction conditions were 25 ° C, 250 rpm, and the induction was 4 h. Then, the bacteria were collected by centrifugation at 12000 rpm for 5 min, which was marked as post-induction samples. The samples before and after induction were resuspended in lysozyme solution, digested at 37°C for 10 min to break the bacteria, and then subjected to SDS-PAGE detection. The theoretical sizes of the screened recombinant humanized filaggrin were 101.52 kDa, 49.67 kDa, 39.60 kDa, 29.58 kDa and 20.75 kDa, respectively.

[0032] The results are as follows Figure 1 as well as Figure 2 As shown, Figure 1Band 1 is the result before PET28a(+)-FLG-1 induction, band 2 is the result after PET28a(+)-FLG-1 induction, band 3 is the result before PET28a(+)-FLG-2 induction, band 4 is the result after PET28a(+)-FLG-2 induction, band 5 is the result before PET28a(+)-FLG-3 induction, and band 6 is the result after PET28a(+)-FLG-3 induction. Figure 2 In the figure, band 7 is the result before PET28a(+)-FLG-4 induction, band 8 is the result after PET28a(+)-FLG-4 induction, band 9 is the result before PET28a(+)-FLG-5 induction, and band 10 is the result after PET28a(+)-FLG-5 induction.

[0033] It can be seen from SDS-PAGE that after induction, obvious bands appear in the middle of 66.2KD-116KD, 45KD-66.2KD, 35KD-45KD, 25KD-35KD and 18.4KD-25KD, respectively, that is, the positive clones screened can express the target protein, and the expression amount is relatively obvious. According to the above experimental results, the series of recombinant humanized filaggrin proteins of the present invention can be successfully induced to express.

[0034] Example 4 Purification of recombinant humanized filaggrin 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 out in Example 2 were fermented in a 250 mL shake flask for 16 h, and the obtained fermentation broth was centrifuged at 10000 rpm for 20 min in a high-speed centrifuge to obtain bacterial sludge, and the bacterial sludge was purified by the following steps to obtain a pure product of recombinant humanized filaggrin: ① Bacterial cell disruption: The bacterial sludge was resuspended in a solution with a mass-to-volume ratio of 1:6 (50 mm PB + 500 mm NaCl, pH = 8.3), and then the bacterial cells were disrupted using a homogenizer with a pressure of 800 bar. The bacterial cells were disrupted 3 times to obtain a bacterial solution containing the target protein; ② Collection of bacterial solution: centrifuge the bacterial solution containing the target protein after the above disruption at 4°C, 20,000 rpm for 20 min, remove the precipitate, and collect the supernatant, which is the bacterial solution containing the target protein; ③ Nickel column purification: The bacterial solution containing the target protein collected in step ② above is purified by nickel column. The purification method is as follows: 1) Nickel column equilibration: First, rinse the column with 3CV of deionized water to remove the protective solution (20% ethanol) in the column, and then rinse the column with 3CV of equilibration buffer (i.e., binding buffer); 2) Loading the bacterial solution: The bacterial solution containing the target protein collected in step ② was passed through a 0.45 μm membrane and then loaded with the sample. The loading volume was 2 CV. 3) Protein removal: Wash the column with washing buffer until the UV280 curve is flat; 4) Elution of target protein: Use elution buffer for elution. Start collecting when a new peak appears at UV280 until the collected peak flattens out. 5) Gel (SDS gel) detection: Take the above target protein elution sample for gel detection. If the detection is qualified (the protein band size is in line with expectations), proceed to the next step; ④ Molecular sieve purification (G25 filler): The target protein that has passed the nickel column purification test in 5) is further purified by the following purification method: 1) Molecular sieve column balance: first rinse the column with 3CV of deionized water to remove the protective solution (20% ethanol) in the column, and then rinse the column with desalting buffer for 3CV; 2) Sample loading: The target protein that has passed the above test is loaded with a sample volume of 0.2CV; 3) Desalting: Continue to use desalting buffer to desalt and rinse the column until the protein flows out and collect the protein.

[0035] Test results such as Figure 3 As shown, Figure 3 In the figure, 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 proteins purified by the above steps is basically greater than 90%.

[0036] Example 5 Cell Adhesion Assay of Recombinant Filaggrin 1. Sample preparation: (1) Preparation of positive control (animal collagen) solution: Prepare animal collagen (using bovine collagen as an example) with a concentration of 1 μg / mL as the positive control, sterilize it with 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. Make two replicate wells. All the above operations are performed under sterile conditions.

[0037] (2) Sample solution preparation: The purified filaggrin obtained in Example 4 was diluted to 1 μg / mL with ultrapure water and two replicate wells were prepared. The above operations were all performed under sterile conditions.

[0038] (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 to fully dissolve, dilute to 1000 mL in a volumetric flask, sterilize, and store in a sealed container at 4°C.

[0039] (4) Preparation of complete culture medium: Take 10 mL of fetal bovine serum and 1 mL of penicillin-streptomycin dual antibody, and add DMEM basal culture medium to make up to 100 mL.

[0040] 2. Test process: The specific operation process is as follows: (1) Take 100 μL of positive control, negative control (ultrapure water) and sample solution and add them to a 96-well plate. Seal and place at 4°C overnight. During the experiment, discard the liquid and wash once with PBS solution for later use. (2) Trypsinize the HaCaT cells in the logarithmic phase, resuspend them in complete medium containing 10% FBS, and add 100 μL of the diluted cell suspension to the above 96-well plate to a cell density of 15,000 cells per well.

[0041] (3) 3 hours after inoculation (can be adjusted according to the cell status), observe the cell adhesion status under a microscope.

[0042] (4) Add 10 μL of CCK8 to each well, mix well and continue incubating for 2 h. Then take it out of the incubator, place it on a microplate reader, detect the absorbance at a wavelength of 450 nm, and record the measurement results.

[0043] (5) The data were processed using Excel software and the results were expressed as adhesion rate. The calculation formula is:

[0044] 3. Test results: Specific test results are as follows: Figure 4 As shown, the results showed that 1 μg / mL recombinant human filaggrin had good cell adhesion promoting activity.

[0045] Example 6 Recombinant human filaggrin cell scratch test 1. Preparation of test sample solution: Dilute bovine collagen and recombinant human filaggrin samples FLG-1, FLG-2, FLG-3, FLG-4, and FLG-5 to 10 μg / mL with serum-free DMEM medium (Gibco, USA), and make two replicate wells. All the above operations were performed under sterile conditions.

[0046] 2. Operation process: (1) First, draw reference lines on the back of the 12-well plate with a marker pen, crossing the center of the wells, with at least 2 lines crossing each well; (2) NIH3T3 cells in the logarithmic phase were digested with trypsin, resuspended in 10% FBS DMEM medium (Gibco, USA), and inoculated into a 12-well plate at a concentration of 150,000 cells / mL and 1 mL / well; (3) After the cells grow to 90% on the second day, dilute the sample to 1 μg / mL with serum-free DMEM medium (Gibco, USA). Then discard the medium in the original cell 12-well plate, leaving 200 μL of medium. Use a 200 μL pipette tip to measure the ruler and scratch as perpendicular to the reference line on the back as possible. The pipette tip should be vertical and not tilted. Wash the cells twice with PBS to remove the detached cells. If there are no floating cells under the microscope, the cells are clean. Then add the prepared sample, 1 mL / well, and mark them. Record the cell scratch data with a microscope (Leica, Germany), record it as 0 hours, and place it in a cell culture incubator (37°C, 5% CO2, Thermo Fisher Scientific, USA) after recording. Select positions for taking pictures at 24 hours and 48 hours (the different time points for each group of pictures are required to be at the same position). The shooting time can be adjusted according to the actual situation; (4) Use ImageJ software to process the images and express the results as mobility. The mobility calculation formula is as follows:

[0047] 3. Test results: The test results are as follows: Figure 5 As shown, the results showed that 1 μg / mL of recombinant human filaggrin had good cell migration promoting activity.

[0048] Example 7 Application of recombinant humanized filaggrin Based on the above experimental results of cell adhesion and migration activity, FLG-2 was selected for the following human efficacy experiment. In order to verify the moisturizing effect of humanized FLG on the skin, that is, the moisturizing effect of recombinant FLG-2 on the skin, this example was verified by the following experiments: 1. Subject Screening Number of subjects: The minimum valid number is 24.

[0049] Inclusion criteria: (1) Healthy male or female aged 18 to 65 years; (2) The base value of the forearm test area measured by a skin moisture tester is between 15 and 45 (Corneometer Unit, CU); (3) The subject's forearm skin is intact, without any scars, injuries or other factors that may affect the assessment; (4) Able to strictly follow the requirements and time schedule of the research plan and sign the informed consent form.

[0050] Exclusion criteria: (1) Women who are currently pregnant, breastfeeding, or planning to become pregnant in the next two months; (2) Patients with severe systemic diseases, immunodeficiency or autoimmune diseases, or patients whose test sites have undergone skin treatment, cosmetic surgery or other tests that may affect the results; (3) Those with active allergic diseases or highly sensitive constitutions; (4) Those who have used hormone drugs and immunosuppressants in the past month; (5) Those who are currently or have participated in other clinical trials in the past three months.

[0051] Exclusion criteria: serious adverse reactions during the test and non-compliance with the test plan (such as using other cosmetics or drugs during the test and requesting to withdraw for other reasons).

[0052] 2. Preparation before the test: The test area cannot be exposed to water for 3 hours before the test. Before the test, the subjects need to clean the flexor side of both forearms, wipe them gently with facial tissue, and sit quietly for 30 minutes in the human efficacy evaluation room (temperature: 21℃±1℃, humidity: 50%±10%). No water or beverages can be consumed during the test. The forearms are exposed and placed in the test state, and remain relaxed. Before the test, the subjects are explained the test and signed the informed consent form.

[0053] The test area is the flexion side of both forearms, divided into five measurement areas: A, B, C, D, and E. Each area is at least 3 cm × 3 cm, and the interval between each test area is at least 1 cm. The inspector selects the test product area, negative control area (using desalting buffer as negative control), and blank control area on the right and left forearms according to the random table. The measurement sites are as follows: Figure 6 shown.

[0054] 3. Product Usage: Based on the regional settings, the inspector uses disposable latex finger cots to evenly apply the test product and negative control product at a dosage of (2.0±0.1) mg / cm² on the designated test area and negative control area. No product is used in the blank control area.

[0055] 4. Objective quantitative evaluation: After wiping the arm with a dry paper towel for 30 minutes, use Delfin MoistureMeter SC to measure and analyze the moisture content of the stratum corneum in all tested areas on the flexor side of the forearm as the initial value (T0h), and then use the product in the selected area. The subject needs to return for a visit 2 hours (T2h) and 4 hours (T4h) after using the product and perform the same indicator test. The test of the same subject is completed using the same instrument and the same tester. The measuring probe should be cleaned between two measurements. The test site should be kept consistent between the two test time points.

[0056] 5. Statistical analysis: Statistic the measurement values ​​of each test area, including quantity, mean, standard deviation, etc.

[0057] Calculate the difference between the initial value of each test area and the measured value at other time points, and then use this difference to statistically analyze the differences between the product area and the negative control area at different time points.

[0058] If the test data is normally distributed, the paired t-test method is used for statistical analysis; if the test data is non-normally distributed, the rank sum test method is used for statistical analysis. The statistical methods all use two-tailed tests, and the test level α=0.05. The statistical data are 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 an extremely significant difference. If there is a significant difference in the values ​​of the various indicators in the test area, it means that the product has a moisturizing effect. When the results show no significant difference, it is marked with ns, indicating that the product does not have a moisturizing effect.

[0059] 6. Test results: (1) Safety: No cases of adverse skin reactions occurred in this trial, indicating that the trial product has good safety.

[0060] (2) Moisturizing effect, specific results are as follows Figure 7-Figure 9 As shown, Figure 7 Descriptive statistics of the stratum corneum water content of the flexor side of the forearm (CU, n=32); Figure 8 Analysis of the difference in water content of the stratum corneum of the flexor side of the forearm (CU, x±s, n=32); Fig. 9 The change of stratum corneum water content before and after use on the flexor skin of the forearm (CU, x ±s, n=32). It can be seen that: 1) Test product (50ppm filaggrin): Analysis of the moisture content of the stratum corneum of the skin showed that after the subjects used the test product (50ppm filaggrin), the moisture content of the stratum corneum of the skin 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 moisture content of the stratum corneum of the skin after using the test product (50ppm filaggrin) for 2 hours and 4 hours was not significantly different from that of the negative control group (p>0.05). The results showed that the test product (50ppm filaggrin) did not have a moisturizing effect at 2h and 4h.

[0061] 2) Test product (100ppm filaggrin): Analysis of the water content of the stratum corneum of the skin showed that after the subjects used the test product (100ppm filaggrin), the water content of the stratum corneum of the skin on the flexor side of the forearm increased significantly at each follow-up time point T2h and T4h (p<0.01), and the difference in the water content of the stratum corneum of the skin 2h and 4h after using the test product (100ppm filaggrin) was significantly different from that of the negative control group (p<0.01).

[0062] 3) Test product (2000ppm filaggrin): Analysis of the moisture content of the stratum corneum of the skin showed that after the subjects used the test product (2000ppm filaggrin), the moisture content of the stratum corneum of the skin on the flexor side of the forearm increased significantly at each follow-up time point T2h and T4h (p<0.01), and the difference in moisture content of the stratum corneum of the skin 2 hours and 4 hours after using the test product (2000ppm filaggrin) was significantly different from that of the negative control group (p<0.01).

[0063] The final results showed that the test products (100ppm and 2000ppm filaggrin) had significant moisturizing effects at 2 hours and 4 hours.

[0064] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope 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 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.

2. A polynucleotide sequence encoding the recombinant humanized filaggrin according to claim 1, characterized in that: 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.

3. A recombinant humanized filaggrin expression vector, characterized in that: It comprises the polynucleotide sequence encoding the recombinant humanized filaggrin as claimed in claim 2.

4. A recombinant engineered bacterium, characterized in that: It comprises the recombinant humanized filaggrin expression vector as described in claim 3.

5. The recombinant engineered bacterium according to claim 4, characterized in that Escherichia coli is used as the host.

6. A method for preparing recombinant humanized filaggrin according to claim 1, characterized in that: The process includes the following: 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 to obtain the target DNA fragments; S2, the target DNA fragment obtained in step S1 was double-digested with XbaI and NcoI and then inserted 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; S3, respectively transferring the recombinant plasmids PET28a-FLG-1 to PET28a-FLG-5 obtained in S2 into Escherichia coli to obtain positive bacteria, further culturing and inducing expression to obtain bacteria containing the recombinant humanized filaggrin FLG; S4, purification: resuspending and breaking the recombinant humanized filaggrin FLG obtained in step S3; The supernatant is collected by centrifugation and filtered, and then the filtered solution is purified by affinity chromatography through a nickel column to obtain the product.

7. The preparation method according to claim 6, characterized in that: The affinity chromatography purification process described in step S4 is as follows: the filtrate containing the recombinant humanized filaggrin FLG is passed through a nickel column, unbound impurities are washed away with binding buffer and washing buffer, and the recombinant humanized filaggrin FLG is eluted with elution buffer; added to a G25 desalting column, and washed out with desalting buffer to obtain high-purity recombinant humanized filaggrin FLG.

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

5.

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

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

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