Recombinant silk fibroin as well as preparation method and application thereof

Abstract

The invention relates to the field of protein engineering, in particular to recombinant silk fibroin as well as a preparation method and application thereof, and an amino acid sequence of the recombinant silk fibroin comprises a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2. The silk fibroin can be expressed in a fungal host, and a basis is provided for developing novel cosmetic raw materials for a biosynthesis platform.

Description

Technical Field

[0001] This invention relates to the field of protein engineering, and in particular to a recombinant silk fibroin, its preparation method, and its uses. Background Technology

[0002] Silk fibroin is a natural high-molecular-weight fibrous protein extracted from silkworm silk, accounting for approximately 70% to 80% of silk. It is the most important component of silk, belonging to the fibrous protein category, and has no significant physiological activity. As a natural material, it possesses excellent biocompatibility, biodegradability, and oxygen permeability.

[0003] Silk fibroin consists of three subunits: a heavy chain (H-chain), a light chain (L-chain), and glycoprotein P25. The heavy and light chains are linked by disulfide bonds, and then further bonded to glycoprotein P25 through non-covalent interactions such as hydrophobic interactions. The heavy chain (H-chain) consists of 5236 amino acid residues with a molecular weight of 391 kDa; the light chain (L-chain) consists of 266 amino acid residues with a molecular weight of 28 kDa; and the P25 protein is similar in size to the light chain, approximately 25 kDa. Because the light chain and P25 protein constitute a very small proportion of the total mass in silk fibroin and are linked to the heavy chain via disulfide bonds and hydrophobic interactions, it is recently believed that the heavy chain, light chain, and glycoprotein P25 assemble into a high-molecular-weight basic structural unit in a 6:6:1 ratio. Therefore, current research and discussion on regenerated silk fibroin used as a material generally refer to the heavy chain or a mixture of heavy and light chains.

[0004] Silk fibroin also has powerful whitening effects, with reported and researched benefits mainly including the following six aspects:

[0005] Moisturizing function: Silk fibroin has extremely strong skin affinity. When it forms a film on the skin surface, it can not only absorb moisture when the external humidity is high, but also release moisture when the external environment is dry, keeping our skin at its optimal hydration level. When the skin barrier is damaged, a natural, non-irritating product with excellent moisturizing properties is needed to repair the skin.

[0006] Anti-wrinkle function: Silk fibroin is known as the "queen of fibers". Its fibrous protein can enhance skin elasticity, accelerate skin metabolism, and prevent the formation of wrinkles.

[0007] Anti-aging function: Collagen is the main reason why skin doesn't age, but once collagen is lost, it cannot regenerate. Silk fibroin's main amino acids are very similar to collagen's, which allows it to accelerate cell metabolism, repair the stratum corneum, delay skin aging, and slow down the aging process.

[0008] Sun protection function: Tryptophan and tyrosine play their biggest role, greatly reducing the damage of ultraviolet rays to the skin. Not only can they protect against the sun, but long-term use can also delay the aging caused by photoaging.

[0009] Whitening function: Silk fibroin can inhibit the formation of melanin in the skin. With less melanin and the added sun protection, the skin naturally becomes whiter and smoother.

[0010] Hair care function: After hydrolysis, silk fibroin becomes silk peptides. Silk peptides have excellent film-forming properties, which can give hair shine and moisturize it. They protect hair from external chemical and heat damage, provide essential nutrients, and moisturize and nourish the hair.

[0011] Currently, the main methods for obtaining silk fibroin are chemical extraction and enzymatic digestion. Chemical extraction is not effective in obtaining large molecular weight peptides. Enzymatic digestion, on the other hand, is limited by enzyme specificity, resulting in impure products. Both methods have poor reproducibility, low batch-to-batch stability, and variations in product quality.

[0012] Genetic engineering techniques can accurately obtain various target proteins at low cost and are environmentally friendly. However, the expression yield of gene sequences encoding silk fibroin in heterologous hosts is limited by their sequence size and highly repetitive fragments. Summary of the Invention

[0013] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a recombinant silk fibroin, its preparation method and uses, to solve the problems in the prior art.

[0014] To achieve the above and other related objectives, the present invention provides a recombinant silk fibroin, wherein the amino acid sequence of the recombinant silk fibroin includes the sequence shown in SEQ ID NO.2 or includes a sequence that has a similarity of more than 95% to the amino acid sequence shown in SEQ ID NO.2 and has the function of recombinant silk fibroin.

[0015] The amino acid sequence of the recombinant silk fibroin is shown in SEQ ID NO.2 or SEQ ID NO.1.

[0016] The present invention also provides an isolated polynucleotide encoding the aforementioned recombinant silk fibroin.

[0017] In some embodiments of the present invention, the isolated polynucleotide comprises a nucleotide sequence as shown in SEQ ID No. 6 or 7.

[0018] The present invention also provides a nucleic acid construct comprising the isolated polynucleotides described above.

[0019] The present invention also provides an engineered cell containing the aforementioned nucleic acid construct or the genome in which exogenous isolated polynucleotides are integrated.

[0020] The present invention also provides a method for preparing the recombinant silk fibroin, comprising the following steps: culturing the engineered cells, inducing the expression of the recombinant silk fibroin, and collecting and separating the bacterial cells after induction to obtain the recombinant silk fibroin.

[0021] The present invention also provides the use of the recombinant silk fibroin, the isolated polynucleotide, the nucleic acid construct or the cell in the preparation of cosmetics or pharmaceuticals.

[0022] The present invention also provides a cosmetic product, the cosmetic product comprising the recombinant silk fibroin and excipients.

[0023] As described above, the recombinant silk fibroin, its preparation method, and its uses according to the present invention have the following beneficial effects: Through genetic engineering, a small-molecule silk fibroin can be recombined and expressed in yeast. The recombinant small-molecule silk fibroin maximally removes redundant portions of the extra structural domains of endogenous silk fibroin contained in the fusion protein, thereby enabling the growth and expression of silk fibroin in fungi and providing a foundation for the development of novel cosmetic raw materials using a biosynthetic platform. Attached Figure Description

[0024] Figure 1 The diagram shown is a schematic representation of the structure of the plasmid pPIC9K-SF-4 of the present invention.

[0025] Figure 2 The image shows a gel image of recombinant silk fibroin fermentation SDS-PAGE protein detection. Line 1: Marker, Line 2: GS115-pPIC9K-SF-4 induced for 96 h, Line 3: GS115-pPIC9K-SF-4 induced for 72 h.

[0026] Figure 3 The image shown is a gel electrophoresis diagram of the PCR product of the recombinant silk fibroin gene SF-10 of the present invention. Line 1: Marker, Line 2: PCR product SF-10 obtained with pPIC9K-SF-4 as template.

[0027] Figure 4 The diagram shows the structure of the recombinant plasmid pPIC9K-SF-10 of the present invention.

[0028] Figure 5The image shown is an SDS-PAG gel image of the recombinant silk fibroin pPIC9K-SF-10 of the present invention. Line 1: pPIC9K-SF-10 induced for 72 h, Line 2: pPIC9K-SF-10 induced for 24 h, Line 3: Marker.

[0029] Figure 6 The results show the effects of the silk fibroin of the present invention on the proliferation of keratinocytes.

[0030] Figure 7 The results show the effect of the silk fibroin of the present invention on the proliferation of fibroblasts.

[0031] Figure 8 The role of silk fibroin in promoting the secretion of type I collagen. Detailed Implementation

[0032] The present invention first provides a recombinant silk fibroin, wherein the amino acid sequence of the recombinant silk fibroin includes the sequence shown in SEQ ID NO.2 or includes a sequence that has a similarity of more than 95% to the amino acid sequence shown in SEQ ID NO.2 and has the function of recombinant silk fibroin.

[0033] The amino acid sequence of the recombinant silk fibroin also includes a sequence having the function of recombinant silk fibroin, formed by substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence shown in SEQ ID NO.2.

[0034] The amino acid sequence of the recombinant silk fibroin is shown in SEQ ID NO.2 or SEQ ID NO.1.

[0035] The present invention also provides an isolated polynucleotide encoding the aforementioned recombinant silk fibroin.

[0036] The polynucleotide can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded.

[0037] The polynucleotide encoding the recombinant silk fibroin of the present invention can be prepared by any suitable technique known to those skilled in the art, such as, but not limited to, recombinant DNA technology, chemical synthesis, etc.

[0038] Based on the already disclosed amino acid sequence of the recombinant silk fibroin, due to the degeneracy of codons, those skilled in the art can obtain the nucleotide sequence of the isolated polynucleotide while keeping its encoding amino acid sequence unchanged. This is a conventional technique in the art.

[0039] In some embodiments of the present invention, the isolated polynucleotide comprises a nucleotide sequence as shown in SEQ ID No. 6 or 7.

[0040] The present invention also provides a nucleic acid construct comprising the isolated polynucleotides described above.

[0041] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector backbone, i.e., an empty vector and an expression framework.

[0042] There is no specific limitation on the type of expression vector. An expression vector is a nucleic acid molecule that allows the insertion of foreign nucleotides without disrupting its ability to replicate and / or integrate into the host cell. Expression vectors may include nucleic acid sequences that allow them to replicate in the host cell, such as origins of replication. Expression vectors may also include one or more selective marker genes and other genetic factors. An expression vector is a vector containing the necessary regulatory sequences to enable the transcription and translation of one or more inserted genes. Expression vectors are selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0043] The prokaryotic expression vector is selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, or Streptomyces expression vectors. In a preferred embodiment, the prokaryotic expression vector is selected from Escherichia coli expression vectors.

[0044] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors. The yeast expression vector is a Pichia pastoris expression vector. The Pichia pastoris expression vector is, for example, any of the following: pPIC9, pPIC9k, pHIL-S1, pPICZa, pYAM75P6, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa. The mammalian expression vector is selected from non-viral vectors or any of the following viral vectors: retroviral expression vectors, lentiviral expression vectors, adenovirus expression vectors, and adeno-associated virus expression vectors.

[0045] The host cell is selected from eukaryotic or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, *C. elegans* or nematodes, or mammalian host cells. Examples of yeast host cells are *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Yarrowia lipolytica*, and *Pichia pastoris*. Examples of prokaryotic host cells include bacterial cells such as *Escherichia coli*, *Streptomyces*, *Bacillus subtilis*, *Salmonella typhi*, or mycobacteria.

[0046] Those skilled in the art can transfect the expression vector into host cells using methods well known in the art to obtain cells containing the encoding gene for recombinant silk fibroin.

[0047] The present invention also provides an engineered cell containing the aforementioned nucleic acid construct or the genome in which exogenous isolated polynucleotides are integrated.

[0048] In some embodiments of the present invention, the engineered cells are yeast. Preferably, the engineered cells are Pichia pastoris.

[0049] The present invention also provides a method for preparing the recombinant silk fibroin, comprising the following steps: culturing the engineered cells, inducing the expression of the recombinant silk fibroin, and collecting and separating the bacterial cells after induction to obtain the recombinant silk fibroin.

[0050] In some embodiments of the present invention, the culture temperature is 28–37.5°C.

[0051] In some embodiments of the present invention, methanol is used to induce aminopeptidase expression. The final volume concentration of methanol is 0.8–1.2 mM, based on the total volume of the reaction system. The induction time is 72–100 hours.

[0052] The recombinant silk fibroin can exist in the form of bacterial fermentation broth or freeze-dried powder, preferably in the form of freeze-dried powder.

[0053] The present invention also provides the use of the recombinant silk fibroin, the isolated polynucleotide, the nucleic acid construct or the cell in the preparation of cosmetics or pharmaceuticals.

[0054] The cosmetics mentioned are selected from cosmetics having one or more of the following functions: anti-aging, anti-wrinkle, skin repair (promoting the repair of damaged skin such as acne scars, sunburn or post-surgery), and anti-inflammatory (reducing skin inflammation and redness, which is beneficial for sensitive skin or inflammatory skin diseases).

[0055] The drug is selected from one or more of the following: wound healing promoting drugs, tissue repair (e.g., eye tissue, periodontal tissue) materials.

[0056] The cosmetics or drugs exert their effects by promoting the secretion of type I collagen, promoting the proliferation of fibroblasts or keratinocytes.

[0057] The present invention also provides a cosmetic product, the cosmetic product comprising the recombinant silk fibroin and excipients.

[0058] The excipients mentioned are those applicable to cosmetics. For example, the excipients comply with the provisions of the "Cosmetic Safety Technical Specifications" or the "Cosmetic Supervision and Administration Regulations".

[0059] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0061] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0062] Example 1: Construction and shake-flask fermentation of silk fibroin strains

[0063] Gene screening:

[0064] Based on the heavy chain (H-chain) of silk fibroin (GenBank: AF226688.1), different silk fibroin sequences were designed using bioinformatics analysis and their nucleotide sequences were integrated into Pichia pastoris for expression. Two expressed sequences, DH5α / pPIC9K-SF-4 and DH5α / pPIC9K-SF-10, were ultimately screened. The screened silk fibroin proteins were purified, and their efficacy was verified. The amino acid sequence of the new protein SF-4 is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.6. The amino acid sequence of SF-10 is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.7.

[0065] SEQ ID No.1(SF-4)

[0066] GAGAGSGAGAGYGAGAGSGAASGAGAGAGATGSSGFGPYVAHGGYSGYEYAWSSESDFGTGS

[0067] SEQ ID No.2(SF-10)

[0068] GAGAGATGSSGFGPYVAHGGYSGYEYAWSSESDFGTGS

[0069] SEQ ID No. 6

[0070] GGAGCAGGAGCGGGCAGTGGAGCGGGAGCGGTATGGTGCTGGAGCTGGAAGCGGCGCCGCCAGTGGAGCAGGAGCGGGTGCAGGAGCTACAGGATCCTCTGGGTTTGGTCCATACGTCGCGCATGGGGCTACAGTGGGTATGAGTATGCGTGGTCCAGCGAGTCGGATTTTGGTACAGGATCA

[0071] SEQ ID No.7

[0072] GGAGCGGGTGCAGGAGCTACAGGATCCTCTGGGTTTGGTCCATACGTCGCGCATGGGGGCCTACAGTGGGTATGAGTATGCGTGGTCCAGCGAGTCGGATTTTGGTACAGGATCA

[0073] (1) Construction and expression of SF-4 strain

[0074] Constructing a host bacterium expressing silk fibroin:

[0075] The SF-4 encoding gene was synthesized by Jiangsu Saisofe Biotechnology Co., Ltd., and cloned into the plasmid pPIC9K (nucleotide sequence shown in SEQ ID NO.3) to construct the recombinant vector pPIC9K-SF-4. A map of the recombinant vector is shown below. Figure 1 The recombinant vectors were linearized by Sac I digestion and then transformed into Pichia pastoris GS115 host. The corresponding engineered strains were obtained after screening with antibiotics.

[0076] SEQ ID NO.3:

[0077]

[0078] The specific operating steps are as follows:

[0079] A. Electroporation of Pichia pastoris cells GS115

[0080] For the electroporation process, add 10 μL of linearized plasmid to a 1.5 mL EP tube containing 80 μL of Pichia pastoris competent cells, mix well, and transfer to a 0.2 cm diameter electroporation cuvette. Incubate the cuvette on ice for 5 min. Electroporation conditions: voltage 1.5 kV; capacitance 25 μF; resistance 200 Ω; electroporation time 4–10 ms. After electroporation, add 650 μL of pre-chilled 1 M sorbitol solution to the electroporation cuvette and gently agitate the solution with a pipette tip. Transfer all liquid from the cuvette to a new 2 mL EP tube and incubate at 30 °C for 2 h. Collect the cells by low-speed centrifugation and spread them onto MD plates (composition shown in Table 1), incubating at 30 °C for 3–4 days.

[0081] Table 1

[0082] materials concentration source Yeast Extract 10g / L Oxoid Tryptone 20g / L Oxoid glucose 20g / L Xi Wang Agar powder 20g / L Haibo

[0083] B. Antibiotic screening

[0084] Once colonies have grown on the plate, use an inoculation loop to pick single bacteria growing on the plate and sequentially transfer them to YPD solid plates containing 0.5 g / L to 4 g / L LG418 antibiotic. Incubate at 30°C for 3 to 4 days.

[0085] C. Small-scale expression identification

[0086] Induction of expression: Single colonies of cells tolerant to 4 g / L G418 were inoculated into 5 mL LYPD medium (composition shown in Table 2) and cultured overnight at 30°C and 220 rpm. The inoculum was then transferred at a rate of 0.01% to a conical flask containing 10 mL BMGY (composition shown in Table 3) and cultured overnight at 30°C and 220 rpm until OD600 reached 2–6 (logarithmic growth, approximately 16–18 h). Cells were collected by centrifugation at 5000 rpm for 5 min at room temperature, the supernatant was removed, and the cells were resuspended in BMMY (composition shown in Table 4) until OD600 reached approximately 1 for induction. Methanol (final volume concentration 1%) was added every 24 h for continued induction, with a total induction time of 96 h. The bands showed a high yield of silk fibroin. The polyacrylamide gel electrophoresis (SDS-PAGE) image is shown below. Figure 2 .

[0087] Table 2

[0088] materials concentration source glucose 20g / L Xi Wang YNB 13.4g / L Lab Biotin 0.0004g / L Adamas Agar powder 20g / L Haibo

[0089] Table 3

[0090] materials concentration source Yeast Extract 10g / L Oxoid Tryptone 20g / L Oxoid Glycerol 20g / L Sinopharm pH 6.0 phosphate 100mM Repackaging YNB 13.4g / L Lab Biotin <![CDATA[4X10 -5 %]]> Adamas

[0091] Table 4

[0092] materials concentration source Yeast Extract 10g / L Oxoid Tryptone 20g / L Oxoid pH 6.0 phosphate 100mM Sinopharm YNB 13.4g / L Lab Biotin 4X10-5％ Adamas methanol 0.5％ Sinopharm

[0093] (2) Construction and shake-flask fermentation of scissor-strain SF-10 containing truncated fibroin

[0094] The recombinant plasmid pPIC9K-SF-4 was transformed into *E. coli* DH5α using the CaCl2 method. Recombinant *E. coli* containing pPIC9K-SF-4 were obtained after screening on LB plates containing Kan, and named DH5α / pPIC9K-SF-4. DH5α / pPIC9K-SF-4 was cultured overnight in liquid LB medium, and plasmid was extracted to obtain a large quantity of pPIC9K-SF-4. PCR was performed using primers containing the pPIC9K homologous arm (SF-10-F and SF-10-R, nucleotide sequences shown in SEQ ID No. 4 and SEQ ID No. 5). The products were subjected to nucleic acid electrophoresis, and the results are shown below. Figure 3 As shown, the target SF-10 fragment was obtained by gel extraction and recovery. The amino acid sequence of SF-10 is shown in SEQ ID NO.2.

[0095] SEQ ID No.4: CTGAAGCTTACGTAGAATTCGGAGCGGGTGCAGG

[0096] SEQ ID No.5: CGAATTAATTCGGCGGCCGCTTATGATCCTGTACC

[0097] The SF-10 target fragment was cloned into pPIC9K to obtain the recombinant plasmid pPIC9K-SF-10, the structure of which is shown in the schematic diagram below. Figure 4 As shown, the cloning method for SF-10 and the construction and shake-flask fermentation method for the Pichia pastoris strain are the same as the construction and shake-flask fermentation steps for the SF-4 silk fibroin strain in this embodiment.

[0098] SDS-PAGE gel image after fermentation is shown below. Figure 5 As shown.

[0099] Example 2: Verification of the efficacy of silk fibroin SF-4

[0100] 1. Verify the effect of silk fibroin on the proliferation of keratinocytes:

[0101] Experimental Objective: To compare the effects of 0.5 g / L silk fibroin, type III collagen, and ergothioneine on keratinocyte proliferation. Background: Keratinocytes are the main cellular component of the human epidermis. These cells proliferate and differentiate, ultimately forming the stratified epidermis, including the basal layer, spinous layer, granular layer, and stratum corneum. Human keratinocytes are important cells in the epidermis and are also UV-sensitive target cells. Recent studies have shown that human keratinocytes play a crucial role in the occurrence and development of photoaging, and are particularly prominent in exacerbating inflammatory responses.

[0102] Experimental principle and scheme:

[0103] MTT assay: Based on the live cell metabolite reducing agent 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazolium romide, MTT thiazolyl blue. MTT is a yellow compound and a hydrogen ion-accepting dye that acts on the respiratory chain in the mitochondria of live cells. Under the action of succinate dehydrogenase and cytochrome C, the tetrazolium ring cleaves, generating blue formazan crystals. The amount of formazan crystals generated is directly proportional to the number of live cells (in dead cells, succinate dehydrogenase is lost and cannot reduce MTT). The reduced formazan crystals can be dissolved in MTT solution containing 50% N,N-dimethylformamide and 20% sodium dodecyl sulfate (pH 4.7). The optical density (OD) value at 490 nm is measured using a microplate reader to reflect the number of live cells.

[0104] (1) Inoculating cells

[0105] Keratinocytes (HaCaT) were seeded at 1,000–10,000 cells per well in 96-well plates, with a volume of 200 μL per well. Silk fibroin, type III collagen, and ergothioneine were then added to a final concentration of 0.5 g / L, respectively. The control group was supplemented with 10% FBS medium.

[0106] (2) Cell culture

[0107] Culture conditions: 95% air, 5% carbon dioxide (CO2), 37℃, culture for 3 days.

[0108] (3) Coloration

[0109] After 3 days of culture, add 20 μL of MTT solution (5 mg / ml prepared with PBS) to each well. Continue incubation for 4 hours, then terminate the culture and carefully aspirate the culture supernatant from the wells. For suspension cells, centrifuge before aspirating the culture supernatant. Add 150 μL of DMSO to each well and shake for 10 minutes to fully dissolve the crystals.

[0110] (4) Color matching

[0111] A wavelength of 490 nm was selected, and the absorbance of each well was measured on an enzyme-linked immunosorbent assay (ELISA) monitor. The results were recorded, and a cell growth curve was plotted with time on the x-axis and absorbance on the y-axis.

[0112] The results are as follows Figure 6 As shown, the silk fibroin of the present invention has a significantly better effect on the proliferation of keratinocytes than collagen, ergothionein and the control group.

[0113] 2. To verify the effect of silk fibroin on fibroblast proliferation.

[0114] Objective: To compare the effects of 0.5 g / L silk fibroin, type III collagen, and ergothionein on fibroblast proliferation.

[0115] Experimental Background: Human skin fibroblasts (HSF) are the main cells in the dermis of human skin, responsible for synthesizing and secreting collagen fibers, elastic fibers, and other organic components. These components, along with the fibroblasts themselves, maintain the elasticity of the dermis. Besides secreting collagen and elastic fibers to maintain skin elasticity, fibroblasts also exhibit chemotaxis and adhesion, playing a crucial role in maintaining skin elasticity and resilience. A decrease in the number of HSFs is a significant cause of wrinkles. Fibroblasts are the main cellular component of the skin, distributed within the dermis. They are the most common cells in loose connective tissue, producing large amounts of collagen and elastic fibers, playing a vital role in maintaining skin structural stability and elasticity. The vitality and proliferation rate of fibroblasts determine skin firmness and thus affect skin condition. Decreased fibroblast vitality and a reduced number of fibroblasts lead to skin aging. The MTT assay can indirectly reflect the number of viable cells and is a universal method for detecting cell activity.

[0116] Experimental procedure:

[0117] (1) Cell seeding: Well-grown primary human fibroblasts were selected, digested, and cultured in cell culture medium to prepare a cell suspension of approximately 10,000 cells / mL. 200 μL of this suspension was seeded into each well of a 96-well plate, and then 0.5 g / L of silk fibroin, 0.5 g / L of type III collagen, and 0.5 g / L of ergothioneine were added to each well. The control group was cultured in DMEM medium containing 2% newborn calf serum. The plates were incubated in a CO2 incubator for 72 h.

[0118] (2) MTT assay: After 72 h of incubation, the culture medium in the culture plate was aspirated, and 200 μL of 0.5 mg / mL MTT solution was added to each well. The plate was incubated in a CO2 incubator for 4 h ± 15 min. After incubation, the MTT solution was aspirated, and 150 μL of LDMSO was added to each well. The 96-well plate was placed on a shaker and shaken in the dark for 10 min. After shaking, the plate was placed in a microplate reader, and the absorbance value (OD value) was read at 490 nm.

[0119] The results are as follows Figure 7 As shown, the silk fibroin of the present invention has a significantly better proliferative effect on fibroblasts than collagen, ergothionein, and the control group.

[0120] 3. Verify the effect of silk fibroin on promoting the secretion of type I collagen.

[0121] Experimental Background: Collagen is one of the most active components of the extracellular matrix, a key factor in wound healing and scar formation, and plays an important role in skin function repair. In normal adult skin, collagen secreted by HSF (human dermal fibroblasts) is mainly of types I and III. Type I collagen constitutes the majority of normal adult skin. Various methods exist for measuring collagen content; ELISA, Western blotting, and immunofluorescence can accurately quantify changes in HSF secretion and collagen content within cultured cells, respectively.

[0122] Experimental Principle: Collagen accounts for approximately 80% of the dermis layer of the skin, making the skin plump and full, and promoting the increase of type I collagen content can help resist wrinkles to some extent. Ultraviolet radiation leads to the production of a large number of free radicals in the skin, further inducing the production of matrix metalloproteinases, thereby promoting collagen degradation. Excessive collagen degradation leads to skin atrophy and wrinkles. Therefore, this experiment uses fibroblasts as the research subject.

[0123] Experimental Objective: To compare the effects of 0.5 g / L silk fibroin, type III collagen, and ergothioneine on promoting type I collagen secretion. Experimental Protocol: Inoculated at an appropriate density (1 × 10⁻⁶). 4 Cells were seeded into 24-well plates and incubated in an incubator (37°C, 5% CO2). When the cell deposition rate in the 24-well plates reached 40%–60%, irradiation was performed at a dose of 9 J / cm². 2 Fibroblasts were used to establish an in vitro photoaging model by overnight UVA irradiation. Cells were cultured with a final concentration of 0.5 g / L of silk fibroin, type III collagen, and ergothioneine, and the cell supernatant was collected. After 24 hours of incubation, the cell culture supernatant was collected in EP tubes. Collagen I assay: The content of type I collagen was detected according to the instructions of the ELISA kit. The results are as follows... Figure 8 As shown, the silk fibroin of the present invention has a significantly better effect on promoting the secretion of type I collagen than type III collagen, ergothioneine and the control group.

[0124] Efficacy verification conclusion: The silk fibroin in this invention has good effects on promoting the secretion of keratinocytes, fibroblasts and type I collagen, providing a research basis for the widespread use of recombinant silk fibroin in the cosmetic field.

[0125] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A recombinant silk fibroin, characterized in that, The amino acid sequence of the recombinant silk fibroin includes the sequence shown in SEQ ID NO.2 or a sequence that has a similarity of more than 95% to the amino acid sequence shown in SEQ ID NO.2 and has the function of recombinant silk fibroin.

2. The recombinant silk fibroin according to claim 1, characterized in that, The amino acid sequence of the recombinant silk fibroin is shown in SEQ ID NO.2 or SEQ ID NO.

1.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the recombinant silk fibroin according to claim 1 or 2.

4. The isolated polynucleotide according to claim 3, characterized in that, The isolated polynucleotides include nucleotide sequences as shown in SEQ ID No. 6 or 7.

5. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the isolated polynucleotide as described in claim 3 or 4.

6. The nucleic acid construct according to claim 5, characterized in that, The nucleic acid construct is a eukaryotic expression vector or a prokaryotic expression vector; preferably, the prokaryotic expression vector is selected from Escherichia coli expression vector, Bacillus subtilis expression vector or Streptomyces expression vector; preferably, the eukaryotic expression vector is selected from yeast expression vector, insect expression vector or mammalian expression vector; more preferably, the yeast expression vector is Pichia pastoris expression vector.

7. An engineered cell, characterized in that, The engineered cells contain the nucleic acid construct of claim 5 or 6 or the genome integrated with exogenous isolated polynucleotides of claim 3 or 4.

8. The engineered cell according to claim 7, characterized in that, The engineered cell is yeast; preferably, the engineered cell is Pichia pastoris.

9. The method for preparing recombinant silk fibroin according to claim 1 or 2, characterized in that, The process includes the following steps: culturing the engineered cells as described in claim 7 or 8, inducing the expression of recombinant silk fibroin, and collecting and separating the bacterial cells after induction to obtain the recombinant silk fibroin.

10. Use of the recombinant silk fibroin of claim 1 or 2, the isolated polynucleotide of claim 3 or 4, the nucleic acid construct of claim 5 or 6, or the engineered cell of claim 7 or 8 in the preparation of cosmetics or pharmaceuticals.

11. The use according to claim 10 or 11, characterized in that, The cosmetic is selected from cosmetics having one or more of the following functions: anti-aging, anti-wrinkle, skin repair, anti-inflammatory; and / or, the drug is selected from one or more of the following: wound healing promoting drugs, tissue repair materials.

12. A cosmetic product, characterized in that, The cosmetic product includes the recombinant silk fibroin as described in claim 1 or 2, as well as excipients.

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