Recombinant keratin polypeptide as well as preparation method and application thereof

Abstract

The invention provides a recombinant keratin polypeptide as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. The amino acid sequence of the recombinant keratin polypeptide is as shown in any one of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7 and SEQ ID No. 10. The recombinant keratin polypeptide has a good soothing effect and the effects of stimulating skin angiogenesis and improving local blood circulation, and can be widely applied to the industries of medicines, cosmetics, skin care products and the like.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a recombinant keratin polypeptide and a preparation method and application thereof. Background Art

[0002] Keratin, as an important cytoskeletal protein, is widely distributed in epithelial and epidermal cells. Its unique biological properties make it an important research object for the development of new biomaterials. Keratin materials have the ability to self-assemble into fibrous nanostructures and exhibit excellent mechanical properties, making them a research hotspot in the field of biomaterials in recent years. In the fields of biomedicine and materials science, keratin materials have been shown to have great potential in many directions.

[0003] Biomaterials developed based on keratin have shown significant application prospects in many fields. For example, in the field of anti-hair loss, keratin materials are used for hair follicle regeneration, dry hair repair and scalp repair; in the field of tissue engineering, its application range covers bones, skin, nerves and blood vessels; in the field of regenerative medicine, keratin materials have shown good effects in bone regeneration, wound healing and nerve regeneration. In addition, in the field of drug delivery, keratin hydrogel has become an ideal carrier choice due to its degradability. It is precisely based on these outstanding performances that keratin materials have received extensive attention and in-depth research in related fields.

[0004] However, there are still some key problems in the practical application of keratin materials. First, naturally extracted keratin needs to undergo severe chemical or physical treatment during the acquisition process. These treatment methods often destroy the natural network structure of keratin, resulting in significant changes in its physical and chemical properties. At the same time, a large number of by-products will inevitably be introduced during these treatments, affecting the purity and performance of the final product. In addition, studies on recombinant keratin have found that even after full-length sequence expression or sequence modification, the co-purification problem of low molecular weight impurities such as melanin and keratin-associated proteins (KAPs) cannot be completely avoided. This leads to problems such as insufficient homogeneity and poor stability in the currently obtained keratin materials.

[0005] The existence of these problems seriously limits the in-depth development and practical application of keratin materials. The instability of material quality directly affects its effect in different application scenarios, making it difficult for related products to achieve ideal performance. At the same time, due to the difficulties in the purification link, the promotion of keratin materials in higher standard applications is also limited. Therefore, how to develop keratin materials that better promote cell proliferation activity and have high homogeneity and good stability is a key problem that needs to be solved in this field. In view of this, the present invention is specially proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant keratin polypeptide and a preparation method and application thereof. The recombinant keratin polypeptide has good soothing effect and the effects of stimulating skin angiogenesis and improving local blood circulation, and can be widely used in the pharmaceutical, cosmetic and skin care products industries.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: In a first aspect, the present invention provides a recombinant keratin polypeptide, the amino acid sequence of the recombinant keratin polypeptide is shown in any one of SEQ ID No.1, SEQ ID No.4, SEQ ID No.7 and SEQ ID No.10.

[0008] In a second aspect, the present invention provides a polynucleotide encoding the recombinant keratin polypeptide as described in the above embodiments.

[0009] In an optional embodiment, the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.12.

[0010] In a third aspect, the present invention provides an expression vector comprising the polynucleotide as described in the aforementioned embodiment.

[0011] In a fourth aspect, the present invention provides a host cell comprising the expression vector described in the aforementioned embodiment.

[0012] In an alternative embodiment, the host cell is Escherichia coli or yeast.

[0013] In a fifth aspect, the present invention provides a method for preparing a recombinant keratin polypeptide as described in the above embodiment, comprising: Cultivating the host cells in a production medium; The recombinant keratin polypeptide is isolated from the host cell.

[0014] In a sixth aspect, the present invention provides a composition comprising the recombinant keratin polypeptide as described in the above embodiments.

[0015] In a seventh aspect, the present invention provides a pharmaceutical product, wherein the pharmaceutical product comprises the composition as described in the above embodiment; In an optional embodiment, the pharmaceutical product is a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic or a health product; In an optional embodiment, the pharmaceutical composition is an external preparation; In an optional embodiment, the topical preparation includes a topical microneedle preparation, a topical hydrogel, or a topical infiltration preparation.

[0016] In an eighth aspect, the present invention provides a use of the recombinant keratin polypeptide as described in the aforementioned embodiment in the preparation of medical devices, tissue engineering products or cosmetics.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a recombinant keratin polypeptide and a preparation method and application thereof, wherein the present invention preferably recombines the helical regions Coil1, Coil2 and the hydrophilic conservative structural region in the human keratin KRT81, and provides four recombinant keratin polypeptide sequences. The recombinant keratin polypeptide can be expressed in Escherichia coli or yeast, and can show better cell proliferation activity than the full-length expressed KRT81 keratin, has a good soothing effect and stimulates skin angiogenesis and improves local blood circulation, and can be widely used in the pharmaceutical, cosmetic and skin care industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a schematic diagram of the preferred sequence of KRT81 in Example 1 of the present application; Figure 2 This is a representative diagram of plasmid construction using vectors pET28a-KRTBERI4 and pPicZalpha-KRTBERI4 as examples in Example 1 of the present application; Figure 3 The protein electrophoresis diagram of the four recombinant keratin polypeptides and KRT81 expressed in E. coli in Example 2 of the present application (the molecular weights of the recombinant proteins KRTBERI4, KRTBERI5, KRTBERI6, and KRTBERI7 detected by electrophoresis are approximately 30 kDa, 30 kDa, 32 kDa, and 20 kDa, respectively; the molecular weight of KRT81 detected by electrophoresis is approximately 50 KDa); Figure 4 The protein electrophoresis diagram of KRTBERI7 expressed in Pichia pastoris in Example 2 of the present application (the molecular weight detected by electrophoresis is approximately 17 kDa); Figure 5This is the protein electrophoresis diagram obtained after the KRTBERI7 protein is expressed and purified in Example 3 of the present application; the electrophoresis detected molecular weight of the KRTBERI7 protein is about 20 kDa; Figure 6 The results of the cell proliferation promoting activity test of the recombinant keratin and the full-length KRT81 expression protein in Example 4 of the present application; Figure 7 This is a representative diagram of the soothing efficacy test of the recombinant keratin and the full-length KRT81 expression protein in Example 5 of the present application; Figure 8 This is a representative diagram of the angiogenesis test of the recombinant keratin and the full-length KRT81 expression protein in Example 6 of the present application. DETAILED DESCRIPTION

[0020] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0021] In an embodiment of the present application, a recombinant keratin polypeptide is provided, and the amino acid sequence of the recombinant keratin polypeptide is shown in any one of SEQ ID No.1, SEQ ID No.4, SEQ ID No.7 and SEQ ID No.10.

[0022] It should be noted that keratin is a type of hard protein belonging to the intermediate filament protein family. It is mainly found in hard or keratinized tissues such as animal epidermal cells, hair, nails, feathers, and horns. Keratin has high mechanical strength and stability and is the main structural protein that constitutes these tissues.

[0023] In the examples of the present application, the recombinant keratin polypeptide provided is derived from the expression region of human keratin KRT81, and preferably, the Coil1, Coil2 and hydrophilic conserved domain sequences in human KRT81 keratin are recombined.

[0024] It should be noted that KRT81 belongs to the "hard" keratin class and is often distributed in epidermal appendages (hair, skin and nails). It is a commonly used protein for synthesizing keratin materials.

[0025] The present invention recombines the helical regions Coil1, Coil2 and the hydrophilic conservative structural region in human keratin KRT81, selects 23-80 consecutive amino acids for multi-fragment recombinant An (n=3-20), and obtains 4 recombinant keratin polypeptides.

[0026] Specifically, the amino acid sequence of the recombinant keratin polypeptide may include any one of SEQ ID No.1, SEQ ID No.4, SEQ ID No.7 and SEQ ID No.10, or may be based on the amino acid sequences of SEQ ID No.1, SEQ ID No.4, SEQ ID No.7 and SEQ ID No.10, and substitution, addition, deletion or insertion operations may be performed on the sequence, and the number of changed amino acid residues is 1 or more, preferably 2, 4, 6 or 8.

[0027] The above-mentioned addition refers to an amino acid addition treatment, which refers to adding amino acids to the C-terminus or N-terminus of any of the four keratin amino acid sequences, as long as the polypeptide has keratin characteristics and cell proliferation promoting activity.

[0028] The substitution mentioned above refers to an amino acid substitution process, which means that an amino acid residue at a certain position in any of the four keratin amino acid sequences is replaced by other amino acid residues, as long as the polypeptide has keratin characteristics and cell proliferation promoting activity.

[0029] As mentioned above, insertion, i.e., amino acid insertion treatment, refers to inserting an amino acid residue at an appropriate position in the sequence of any one of the four keratin amino acid sequences. The inserted amino acid residues may be adjacent to each other in whole or in part, or the inserted amino acids may not be adjacent to each other, as long as the polypeptide has keratin characteristics and cell proliferation promoting activity.

[0030] The above, indeed, the amino acid deletion treatment, refers to deleting 1, 2 or 3 or more amino acids in the sequence of any one of the four keratin amino acid sequences, as long as the polypeptide has keratin characteristics and cell proliferation promoting activity.

[0031] It should be noted that the substitution for the recombinant keratin polypeptide in the examples of the present application may be a conservative amino acid substitution, which means that compared with the amino acid sequence of any of the four keratin amino acid sequences, three, preferably two or one amino acid is replaced by an amino acid with similar or similar properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 1.

[0032] A polynucleotide is provided in the examples of the present application, wherein the polynucleotide encodes the recombinant keratin polypeptide as described in the above embodiments.

[0033] In an optional embodiment, the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.12.

[0034] An expression vector is provided in the examples of the present application, comprising the polynucleotide as described in the above embodiments. An expression vector is provided in the examples of the present application, comprising the expression vector as described in the above embodiments. In an optional embodiment, the host cell is Escherichia coli or yeast.

[0035] The present application provides a method for preparing the recombinant keratin polypeptide as described in the above embodiment, comprising: Step S1, culturing host cells in a production medium.

[0036] It should be noted that the current research on keratin materials is mainly focused on the extraction and processing technology of natural keratin. Traditional extraction methods mostly use high temperature, high pressure or strong acid and alkali treatment. This drastic chemical and physical treatment method not only easily destroys the natural structure of keratin, but also may lead to changes in the assembly of keratin networks and the generation of a large number of by-products. These by-products can affect the biological characteristics and performance of keratin in some cases, limiting its application in some sensitive fields (such as skin repair and regenerative medicine). In order to overcome these problems, some researchers have tried to use genetic engineering technology to produce keratin using transgenic cells or recombinant protein expression systems. The keratin materials obtained in this way can maintain a high stability in structure and function.

[0037] However, existing genetic engineering technologies still face certain challenges, especially in the purification process of recombinant keratin. Studies have found that although recombinant keratin can be effectively obtained by expressing the full-length sequence of keratin genes such as KRT81, or by replacing and modifying their sequences with amino acids, this recombinant protein will still be accompanied by low-molecular-weight impurities such as melanin and keratin-associated proteins (KAPs). These impurities not only increase the difficulty of purifying recombinant keratin, but may also have a negative impact on the biological activity of keratin and the final material properties. Existing purification technologies still find it difficult to efficiently remove these impurities, resulting in less than ideal purity and uniformity.

[0038] In summary, the development and application of existing keratin materials have many problems, such as structural damage caused by the extraction method, difficulty in removing impurities during the purification process, and insufficient stability and functionality of recombinant proteins. These technical defects have, to a certain extent, restricted the in-depth application of keratin in a wider range of fields, especially in the field of high-end biomedical materials that require high keratin purity and functionality.

[0039] Therefore, a method for preparing a recombinant keratin polypeptide is provided in the embodiments of the present application to solve the above-mentioned defects in a targeted manner.

[0040] In the above, the host cell may be the host cell described in the aforementioned embodiment.

[0041] This step may specifically include: (1) Construction of genetically engineered Escherichia coli and yeast.

[0042] (2) Fermentation culture of genetically engineered Escherichia coli and yeast to obtain host cells.

[0043] The above steps may involve selecting host cells and preparing culture medium, then establishing culture conditions and conducting culture, inoculating the host cells into a culture medium containing corresponding antibiotics, conducting shake flask culture, obtaining host cell culture fluid, inoculating the culture fluid and culture medium into a culture medium containing corresponding antibiotics, continuing shake flask culture, and then adding an inducer to induce expression.

[0044] Step S2, isolating and obtaining the recombinant keratin polypeptide from the host cell.

[0045] For example, the above step S2 may specifically include: (3) Inducing and expressing the recombinant keratin polypeptide in the obtained host cells; (4) Purification of recombinant keratin peptides.

[0046] After fermentation is completed, the bacteria are collected by centrifugation or tangential flow filtration system. Centrifugation is fast and efficient, and is suitable for large-scale production. The collected bacteria need to be lysed to release the recombinant keratin polypeptide. Lysis methods include ultrasound, homogenization, high pressure, hypotonicity, lyase, organic solvents, etc. During the lysis process, the temperature, pH value, time and pressure conditions need to be controlled to avoid protein inactivation or degradation. After the lysis is completed, the feed liquid is separated and clarified by centrifugation equipment combined with a tangential flow microfiltration system, and the supernatant is collected.

[0047] In the above purification process, the following purification methods may be included: Preliminary purification: separation and preliminary purification of recombinant keratin polypeptides by multi-step chromatography (such as affinity chromatography, ion exchange chromatography, size exclusion chromatography). High-pressure chromatography purification: further purification of recombinant keratin polypeptides by reverse phase high performance liquid chromatography (RP-HPLC) to obtain a high-purity target protein. Post-purification treatment: the purified recombinant keratin polypeptide solution can be freeze-dried to obtain a powdered product.

[0048] Through the above detailed steps, efficient production, separation and purification of recombinant keratin peptides can be achieved. These technical details and optimization steps help to improve the yield and purity of recombinant keratin peptides to meet the needs of biomedicine and other fields.

[0049] A composition is provided in an embodiment of the present application, comprising the recombinant keratin polypeptide as described in the above embodiment.

[0050] A pharmaceutical product is provided in an embodiment of the present application, wherein the pharmaceutical product comprises the composition as described in the above embodiment; In an optional embodiment, the pharmaceutical product is a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic or a health product; In an optional embodiment, the pharmaceutical composition is an external preparation; In an optional embodiment, the topical preparation includes a topical microneedle preparation, a topical hydrogel, or a topical infiltration preparation.

[0051] The present application provides an application of the recombinant keratin polypeptide as described in the above-mentioned embodiment in the preparation of medical devices, tissue engineering products or cosmetics.

[0052] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0053] The test materials and reagents used in the examples are as follows: 1. Strains, cells and vectors: Strains containing target genes and expression plasmids, Escherichia coli DH5a, TOP10, DE3, Rosetta (DE3), BL21 (DE3) PLysE, BL21 (DE3) PLysS and other strains were purchased from Bio-Tech and Pichia pastoris X33 (Miaoling Biotechnology), and vectors pet28a, pPICzalpha and antibiotic Zeocin were purchased from Invitrogen.

[0054] 2. Kits and enzymes: LDH detection kit (Roche 04744926001), modified Bradford protein content assay kit (Sangon Biotechnology), seamless cloning kit In-Fusion Snap Assembly Master Mix (TaKaRa), plasmid extraction kit (Universal Biotechnology), universal gel recovery kit (Universal Biotechnology), restriction endonuclease, Q5 ultra-fidelity 2x Master Mix PCR polymerase, etc. were purchased from NEB.

[0055] 3. Culture medium: The culture medium for Escherichia coli is LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0), LB+AMP medium is LB medium with ampicillin added to a final concentration of 100 ug / mL, LB+Kana medium is LB medium with kanamycin added to a final concentration of 25 ug / mL, and LB+Zeo is LB medium with Zeocin added to 100 ug / mL.

[0056] The induction medium for Escherichia coli was TB medium (2% yeast powder, 1% peptone, 1.5% dipotassium hydrogen phosphate, 2% potassium dihydrogen phosphate, adjusted to pH 7.0).

[0057] The medium for high-density fermentation of large intestine is TB with 0.1%-0.2% defoamer added, pH is 7.0, and the feed glycerol is 400g / L.

[0058] Yeast culture medium includes YPG medium (1% yeast extract, 2% peptone, 1% glycerol) and YPD medium (1% yeast extract, 2% peptone, 2% glucose). YPG+Zeo medium is YPG medium with 100ug / mL Zeocin added.

[0059] YPD medium is YPD medium with 100 ug / mL of Zeocin added.

[0060] Yeast induction medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (v / v)) and BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 0.5% methanol (v / v)).

[0061] The fermentation medium for recombinant yeast was BSM medium (0.5% potassium dihydrogen phosphate, 5% diammonium hydrogen phosphate, 1.5% magnesium sulfate 7 hydrate, 0.1% calcium sulfate, 2% potassium sulfate, 0.03% defoamer). After high pressure, 4.35 mL of trace salt solution PTM1 was added to the BSM medium, wherein the composition of PTM1 was: 6.5% iron sulfate 7 hydrate, 0.5% copper sulfate, 0.3% manganese sulfate, 0.05% cobalt chloride hexahydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.018% potassium iodide, 2% zinc chloride, 0.5% concentrated sulfuric acid, and 0.02% biotin.

[0062] 4. Protein purification materials: Ni-NTA filler and nickel column were purchased from Sanji Biotechnology.

[0063] 5. Skin soothing experimental materials: SymCalmin® (143535) raw materials are purchased from Symrise.

[0064] Example 1: Gene synthesis of recombinant keratin polypeptides Experimental methods: (1) Gene analysis: The amino acid sequence of natural human hair keratin KRT81 (Genebank: AAH06452.1) was selected for analysis of physical and chemical properties such as hydrophilicity and isoelectric point. The helical regions Coil1, Coil2 and the hydrophilic conservative structural regions in human keratin KRT81 were selected for recombining. Figure 1 shown.

[0065] The recombinant sequence was codon optimized for Escherichia coli and Pichia pastoris, and the new genes obtained by recombinant were genes KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7 and KRT81 in this example, with amino acid sequences such as SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, and SEQ ID NO.13.

[0066] (2) Gene synthesis: The full lengths of KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7 and KRT81 genes are 687 bp, 735 bp, 647 bp, 450 bp and 1518 bp, respectively. The sequences after codon optimization are: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.14 and SEQ ID NO.15.

[0067] The optimized sequence was subjected to gene synthesis, and the gene fragments were provided by General Gene Biotechnology Co., Ltd. The synthesized KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7 and KRT81 gene fragments were inserted into the expression vector of PET28a or PET24a through BamHI and HindIII, NcoI and XhoI, respectively, and then the vector was transformed into Escherichia coli TOP10 or DH5a for preservation.

[0068] The above-mentioned vector maps represented by the KRTBERI4 gene are shown in Figure 2 .

[0069] Example 2: Construction and induced expression of recombinant keratin polypeptides Experimental methods: 1. Construction of genetically engineered Escherichia coli: (1) Plasmid extraction: Referring to Example 1, different strains containing the target gene were streaked and inoculated on LB+A or LB+K plates containing the corresponding resistance, and cultured overnight at 37°C. Subsequently, monoclonal colonies were selected from the plates cultured overnight, inoculated into 10 mL of LB+A or LB+K liquid culture medium, and continued to be cultured overnight at 37°C. Take an appropriate amount of overnight cultured bacterial solution, centrifuge at 5000 rpm for 5 minutes, remove the supernatant to collect the bacterial precipitate. Use the Tiangen Plasmid Extraction Kit to extract the plasmid. The concentration of the extracted plasmid is measured by NanoDrop, and it is directly used to transform the expression host or stored at minus 20°C as needed.

[0070] (2) Strain construction: Take 2 μL of the plasmid containing the target gene and add it to 100 μL of E. coli competent cells, and let it stand on ice for 15-30 min. Heat shock the mixture in a 42℃ water bath for 45s, let it stand on ice for 2-3min, add 500 μL of LB liquid medium without resistance, and culture it at 37℃, 190 rpm for 20 min. Take 100 μL of the bacterial solution and evenly spread it on LB+K or LB+Amp plates. Culture it at 37℃ overnight until clearly visible colonies grow.

[0071] (3) Induced expression of target protein: From the overnight culture plate, pick a single clone and culture it overnight in 10 mL LB+A or LB+K liquid medium. Then transfer it to TB medium at a ratio of 1% for expansion culture. When the OD600 of the bacterial solution is between 0.5 and 0.8 at 37°C and 190 rpm, add IPTG with a final concentration of 0.1 to 0.5 mM to induce expression. The induction conditions are 16-26°C and 190 rpm for 16-20 h. Collect the bacteria by centrifugation and wash them repeatedly with phosphate buffer for 3 times. The washed bacteria can be frozen at minus 20°C or directly lysed with a high-pressure lysing instrument, centrifuged at 10,000 rpm for 30 min, and the supernatant after centrifugation is stored.

[0072] (4) SDS-PAGE detection of target protein: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, place in 100℃ boiling water for 10 min, then add 20 μL per well to the SDS-PAGE protein gel, first run at 80 V for 1 h, then turn to 120 V and run until the bands are completely separated. Use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) to stain the protein for 30 min, and then use protein destaining solution (10% acetic acid, 5% ethanol) to destain overnight.

[0073] Results Figure 3 The electrophoresis detected molecular weights of proteins of genes such as KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7 and KRT81 were approximately 30 kDa, 30 kDa, 32 kDa, 20 kDa and 50 kDa, respectively.

[0074] 2. Construction of genetically engineered yeast: (1) Plasmid extraction: Take different strains containing the target gene in Example 1 and streak them on LB+Z plates with corresponding resistance, and culture them at 37°C overnight. Pick single clones cultured overnight and inoculate them into 10 mL LB+Z liquid culture medium, and culture them at 37°C overnight. Take the bacterial solution cultured overnight, centrifuge at 5000 rpm for 5 min, discard the supernatant and take the bacterial cells. Use Tiangen Plasmid Extraction Kit to extract plasmids. The extracted plasmids are measured by NanoDrop and then frozen at -20°C.

[0075] (2) Plasmid linearization and recovery: Take 20 ug of the extracted plasmid, digest with PmeI at 37°C for 3-6h, take 5 μL of the linearized gene fragment, and detect it with 1% agarose gel to confirm that the fragment is completely linearized. Use Tiangen Universal DNA Purification and Recovery Kit for liquid recovery. The specific steps are as follows: Take the completely linearized mixed solution, add an equal volume of PC solution, mix well and transfer to the collection column after BL equilibration, centrifuge at 12000 rpm for 1 min, rinse with PW solution twice, and centrifuge at 12000 rpm for 2 min. Transfer the collection column to a clean 1.5 mL centrifuge tube, dry at room temperature for 5 min, add ddH2O preheated at 55°C, let stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain the linearized gene fragment.

[0076] (3) Strain construction: Take 10 μL of the linearized fragment and add 100 μL of Pichia pastoris competent medium, mix well on ice and transfer to an electrode cup, and electroshock at 150 V and 200 mA for 10 ms. Immediately after the electroshock, add 500 μL of recovery medium, mix well and transfer to a 1.5 mL centrifuge tube, and let it stand in a 30°C incubator for 3 h to recover. Take 100 μL and spread on a YPG+Z plate, and incubate it upside down in a 30°C incubator for 2-3 days.

[0077] (4) Induction culture: Add BMGY to a 24-well plate, pick the yeast from the cultured plate and transfer it to the 24-well plate, shake and culture in a shaker at 30°C for 1 day, add 100 μL BMMY culture medium for induction, and continue induction culture for two days. At the end of the culture, centrifuge at 4000 rpm for 5 min, and save the supernatant or directly perform SDS-PAGE.

[0078] (5) Protein expression detection: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, place in 100℃ boiling water for 10 min, then add 20 μL per well to SDS-PAGE protein gel, first run at 80 V for 1 h, then turn to 120 V until the bands are completely separated. Use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) to stain the protein for 30 min, and then use protein destaining solution (10% acetic acid, 5% ethanol) to destain overnight.

[0079] Results Figure 4 The molecular weight of KRTBERI7 detected by protein electrophoresis was approximately 17 kDa.

[0080] Example 3: Purification of recombinant keratin polypeptides Experimental methods: (1) Crude protein treatment: Take the frozen cells washed with phosphate buffer, weigh them, resuspend them in 8 volumes of lysis buffer (50 mM PB, 10 mM imidazole, 0.5 M NaCl, pH 7.0), and break them using a high-pressure homogenizer at low temperature. Centrifuge the mixture at 4°C, 10,000 rpm for 25 min, discard the precipitate, harvest the supernatant, and measure the volume.

[0081] (2) Protein purification using Ni-NTA nickel column: Use (50 mM PB, 0.5 M NaCl, pH 7.0) to wash 5 column volumes to balance the nickel column: Load the pretreated protein. Wash with low concentration imidazole (10-50 mM) buffer for 3-5 column volumes to remove impurities, and then perform gradient elution with 50-500 mM imidazole.

[0082] The protein samples eluted with different concentrations of imidazole were collected and the purity was tested by SDS-PAGE. After the collection was completed, the nickel column was washed with 20% ethanol and stored after being filled with ethanol.

[0083] Nickel columns that have been used repeatedly need to be regenerated. The regeneration steps are as follows: rinse with 5 column volumes using ddH2O, rinse with 5 column volumes using EDTA solution, rinse with 5 column volumes using NaOH solution, fill the affinity column with NiSO4 solution, and rinse with 5 column volumes.

[0084] Take the protein purification solutions of different receiving levels and perform SDS-PAGE detection to determine the purity and size of the target protein.

[0085] Results Figure 5 The molecular weight of KRTBERI7 detected by protein electrophoresis was approximately 20 kDa.

[0086] Example 4: Test of the cell proliferation promoting activity of recombinant keratin Experimental methods: (1) L929 cell culture: L929 cells were placed in DMEM medium containing 10% fetal bovine serum and double antibody, and cultured in a carbon dioxide incubator at 5% concentration, 95% relative humidity, and 37°C. The culture medium was replaced every two days, and the cells were passaged after they were basically confluent on the fifth day.

[0087] First, remove the old culture medium, wash twice with PBS solution, and then add 0.5 mL of 0.25% trypsin for digestion. Observe under an inverted microscope, and when the cells shrink and become round, add 10 mL of DMEM culture medium to stop digestion.

[0088] Gently pipette the cells to suspend them, centrifuge at 1000 r / min for 5 min, remove the supernatant, add an appropriate amount of serum-containing culture medium, pipette gently again to make a cell suspension, and take 50 μL for counting.

[0089] For L929 cells in the logarithmic growth phase, the upper medium was removed, the cells were washed twice with PBS solution, digested into a single cell suspension using 0.5 mL of 5% DMEM-0.25% trypsin, and washed twice again with PBS solution to directly collect the suspended cells.

[0090] (2) MTT assay: Select a 96-well plate and inoculate 2×10 4 The experimental group, blank control group (containing only PBS solution) and calibration zero adjustment group (containing no cells) were set up, and two replicate wells were set up for each group.

[0091] After 24 h, most of the cells were found to have attached to the wall and expanded under the microscope. At this time, the cells and liquid that were not attached to the wall were removed. The peptide concentration in the experimental group was adjusted to 0.5 mg / mL, and 20 μL was added, while the blank control group was added with 20 μL PBS buffer, and the correction and zeroing group was added with only DMEM medium.

[0092] When adding samples, make sure the tip of the pipette is close to the wall of the well and inject slowly. After adding each well, gently blow to mix. Then let it stand for 30 minutes and put the plate in a CO2 incubator. Observe the proliferation of cells after 72 hours of culture. Use the MTT method to determine the absorbance (OD) value. Add 20 μL of MTT solution to each well of the 96-well plate and continue to culture for 4 hours to complete the color reaction. After that, terminate the culture, aspirate the culture medium in the well or cover the culture plate with a layer of filter paper, quickly turn the culture plate over, add 150 μL of DMSO to each well, shake for 10 minutes to fully dissolve the formazan, and finally use an enzyme reader to measure the absorbance of each well at a wavelength of A490 nm.

[0093] Experimental results: Table 1. Cell proliferation activity detection data of recombinant keratin and KRT81 full-length expression protein in this example

[0094] Results Figure 6 As shown in Table 1, compared with blank and full-length expressed KRT81 proteins, recombinant keratin proteins KRTBERI4, KRTBERI5, KRTBERI6 and KRTBERI7 have better effects of promoting proliferation and differentiation of L929 cells and enhancing cell activity. The human recombinant keratin of the present invention has the effect of promoting cell proliferation and can be used to prepare biomaterials or drugs that promote cell proliferation.

[0095] Example 5: Test of the cell proliferation promoting activity of recombinant keratin In this example, in order to confirm whether the recombinant keratin polypeptide has the effect of soothing the skin, histamine was used to model the skin inflammation and edema stimulation, and different experimental samples were applied after modeling to test the soothing effect of the recombinant keratin polypeptide on human skin. The specific steps are as follows: (1) Inflammation modeling: Before the experiment, the subjects need to clean the flexed side of both forearms with a dry paper towel, expose the flexed side of the forearms, and place them in the test state, keep relaxed, and avoid touching the test area. The test area is the flexed side of both forearms, which is divided into four measurement areas: A, B, C, and D. The area of ​​each area is 16 square centimeters (4cm × 4cm). Use Antera 3D skin imaging measuring instrument to take pictures of the forearms and measure the skin redness values ​​of each test area. The measured values ​​are used as the data before modeling. In the designated area, apply low-sensitivity medical tape to the middle of the test area. After pressing, tear the tape off the skin from one end, and repeat 10 times. Then use a 3*3cm non-woven fabric to completely soak the 1% histamine solution and apply it to the test area of ​​both forearms of the subjects for 10 minutes. Remove the non-woven fabric, wait until the surface is dry and there is no solution residue, and use Antera 3D skin imaging measuring instrument to take pictures and measure the skin redness values ​​at the center of each test area. The measured values ​​are used as the T modeling data after modeling.

[0096] (2) Sample testing: After the T modeling data is collected, the samples are applied for soothing effect testing, and blank (no treatment), negative control (placebo applied after modeling), recombinant keratin (recombinant keratin applied after modeling) and positive control (Deminshu applied after modeling) are set up. The subjects need to undergo the same index test 15 minutes, 1 hour and 2 hours (T15min, T60min and T120min) after using the product. If the change results of any parameter in the instrument test parameters before and after any visit of the test group (side) using the product are significantly different (P<0.05), or the test value results after using the sample are significantly better than the control group (side) results (P<0.05), then the test product is considered to have the corresponding efficacy, otherwise it is considered that the test product has no corresponding efficacy.

[0097] Results Figure 7 Compared with the control group, the four recombinant keratin polypeptides of the present invention all have better soothing effects.

[0098] Example 6: Skin capillary regeneration test In this example, in order to study whether the recombinant keratin polypeptide has an effect on skin angiogenesis to improve local skin circulation, male mice were depilated and 5% recombinant keratin polypeptide solution and blank control (5% ethanol) were applied topically every day, with 8 mice in each group. The mice were killed on the 15th day of the experiment, and the skin of the back test area was peeled and sliced. The skin slices were immersed in physiological saline, and the skin blood vessels were observed with the assistance of a Nikon SMZ18 stereo microscope.

[0099] The results are as follows Figure 8 As shown, the use of 5% recombinant keratin polypeptide solution can significantly increase the number and density of capillaries in the back skin of mice compared with the blank group, indicating that the recombinant keratin polypeptides in the present invention have the effect of promoting capillary regeneration and improving local circulation.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant keratin polypeptide, characterized in that: The amino acid sequence of the recombinant keratin polypeptide is shown in any one of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7 and SEQ ID No.

10.

2. A polynucleotide, characterized in that The polynucleotide encodes the recombinant keratin polypeptide according to claim 1.

3. The polynucleotide according to claim 2, characterized in that The nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.

12.

4. An expression vector, characterized in that: Comprising the polynucleotide as claimed in claim 2.

5. A host cell, characterized in that Comprising the expression vector as claimed in claim 4.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli or yeast.

7. A method for preparing the recombinant keratin polypeptide according to claim 1, characterized in that: include: Cultivating the host cells in a production medium; The recombinant keratin polypeptide is isolated from the host cell.

8. A composition, characterized in that The invention comprises the recombinant keratin polypeptide as claimed in claim 1.

9. A pharmaceutical product, characterized in that The pharmaceutical product comprises the composition according to claim 8.

10. The pharmaceutical product according to claim 9, characterized in that The pharmaceutical product is a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic or a health product.

11. The pharmaceutical product according to claim 10, characterized in that The pharmaceutical composition is an external preparation.

12. The pharmaceutical product according to claim 11, characterized in that The topical preparation includes a topical microneedle preparation, a topical hydrogel or a topical infiltration preparation.

13. Use of the recombinant keratin polypeptide according to claim 1 in the preparation of medical devices, tissue engineering products or cosmetics.

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