A recombinant keratin polypeptide, its preparation method and application

Abstract

The present invention provides a recombinant keratin polypeptide, a preparation method and an application thereof, relating to the technical field of genetic engineering. The amino acid sequence of the recombinant keratin polypeptide is shown as 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 good soothing effects and the efficacy of stimulating skin angiogenesis and improving local blood circulation, and can be widely applied in the pharmaceutical industry.

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 object of the present invention is to provide a recombinant keratin polypeptide, a preparation method thereof and an application thereof. The recombinant keratin polypeptide has good soothing effects and the efficacy of stimulating cutaneous angiogenesis and improving local blood circulation, and can be widely applied in the pharmaceutical industry.

[0007] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0008] In the first aspect, the present invention provides a recombinant keratin polypeptide, and the amino acid sequence of the recombinant keratin polypeptide is shown as any one of SEQ ID No.1, SEQ ID No.4, SEQ ID No.7 and SEQ ID No.10.

[0009] In the second aspect, the present invention provides a polynucleotide, and the polynucleotide encodes the recombinant keratin polypeptide as described in the foregoing embodiment.

[0010] In an optional embodiment, the nucleotide sequence of the polynucleotide is shown as any one of SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11 and SEQ IDNO.12.

[0011] In the third aspect, the present invention provides an expression vector, which comprises the polynucleotide as described in the foregoing embodiment.

[0012] In the fourth aspect, the present invention provides a host cell, which comprises the expression vector as described in the foregoing embodiment.

[0013] In an optional embodiment, the host cell is Escherichia coli or yeast.

[0014] In the fifth aspect, the present invention provides a preparation method of the recombinant keratin polypeptide as described in the foregoing embodiment, comprising:

[0015] culturing to obtain a host cell in a production medium;

[0016] isolating the recombinant keratin polypeptide from the host cell.

[0017] In the sixth aspect, the present invention provides a composition, which comprises the recombinant keratin polypeptide as described in the foregoing embodiment.

[0018] In the seventh aspect, the present invention provides a pharmaceutical product, and the pharmaceutical product contains the composition as described in the foregoing embodiment;

[0019] In an optional embodiment, the pharmaceutical product is a pharmaceutical composition, a medical device or a tissue engineering product;

[0020] In an alternative embodiment, the pharmaceutical composition is a topical preparation;

[0021] In an alternative embodiment, the topical preparation includes a topical microneedle preparation, a topical hydrogel preparation or a topical infiltration preparation.

[0022] In an eighth aspect, the present invention provides an application of the recombinant keratin polypeptide as described in the foregoing embodiments in the preparation of medical equipment supplies or tissue engineering products.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The present invention provides a recombinant keratin polypeptide, a preparation method and an application thereof. Among them, the present invention preferably recombines the Coil1, Coil2 and hydrophilic conserved structural regions in human keratin KRT81, and provides 4 recombinant keratin polypeptide sequences. The recombinant keratin polypeptide can be expressed in Escherichia coli or yeast, and compared with the full-length expressed KRT81 keratin, it can show better cell proliferation promoting activity, has good soothing effects and the efficacy of stimulating skin angiogenesis and improving local blood circulation, and can be widely applied in the pharmaceutical industry. Brief Description of the Drawings

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 It is a schematic diagram of the preferred sequence of KRT81 in Example 1 of the present application;

[0027] Figure 2 It is a representative diagram of plasmid construction taking the vectors pET28a-KRTBERI4 and pPicZalpha-KRTBERI4 as examples in Example 1 of the present application;

[0028] Figure 3 It is a protein electrophoresis diagram of 4 recombinant keratin polypeptides and KRT81 expressed in Escherichia coli in Example 2 of the present application (the electrophoretic detection molecular weights of the recombinant proteins KRTBERI4, KRTBERI5, KRTBERI6, and KRTBERI7 are approximately 30 kDa, 30 kDa, 32 kDa, and 20 kDa respectively; the electrophoretic detection molecular weight of KRT81 is approximately 50 KDa);

[0029] Figure 4This is the protein electrophoresis pattern of KRTBERI7 expressed in Pichia pastoris in Example 2 of this application (the molecular weights detected by electrophoresis are approximately 17 kDa);

[0030] Figure 5 This is the protein electrophoresis pattern of the purified KRTBERI7 protein obtained in Example 3 of this application; the molecular weight of the KRTBERI7 protein detected by electrophoresis is approximately 20 kDa;

[0031] Figure 6 This is the detection result of the cell proliferation promoting activity of the recombinant keratin and the full-length expressed protein of KRT81 in Example 4 of this application;

[0032] Figure 7 This is the representative diagram of the soothing effect test of the recombinant keratin and the full-length expressed protein of KRT81 in Example 5 of this application;

[0033] Figure 8 This is the representative diagram of the angiogenesis test of the recombinant keratin and the full-length expressed protein of KRT81 in Example 6 of this application. Detailed implementation mode

[0034] The following will describe the implementation plan of the present invention in detail in combination with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified for the manufacturer can be obtained as conventional products through commercial purchase.

[0035] In the examples of this 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.

[0036] It should be noted that keratin is a type of scleroprotein and belongs to the intermediate filament protein family. It is mainly present in the hard or keratinized tissues such as the epidermal cells, hair, nails, feathers, and horns of animals. Keratin has high mechanical strength and stability and is the main structural protein that constitutes these tissues.

[0037] In the examples of this application, the provided recombinant keratin polypeptide is from the expression region of human keratin KRT81, and preferably, the Coil1, Coil2, and hydrophilic conserved domain sequences in human KRT81 keratin are recombined.

[0038] 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.

[0039] The present invention recombines the helical regions Coil1, Coil2 and hydrophilic conserved structural regions in human keratin KRT81, selects 23 - 80 consecutive amino acids for multi - fragment recombination An (n = 3 - 20), and obtains 4 recombinant keratin polypeptides.

[0040] 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 are performed on their sequences, and the number of changed amino acid residues is 1 or more, preferably 2, 4, 6 or 8.

[0041] The above - mentioned "addition" refers to amino acid addition treatment, which means adding amino acids to the C - terminal or N - terminal of any one of the 4 keratin amino acid sequences, as long as the polypeptide has keratin characteristics and cell - proliferation - promoting activity.

[0042] The above - mentioned "substitution" refers to amino acid substitution treatment, which means that a certain amino acid residue at a certain position in any one of the 4 keratin amino acid sequences is replaced by another amino acid residue, as long as the polypeptide has keratin characteristics and cell - proliferation - promoting activity.

[0043] The above - mentioned "insertion", that is, amino acid insertion treatment, means inserting amino acid residues at appropriate positions in the sequence of any one of the 4 keratin amino acid sequences. The inserted amino acid residues may all or partially be adjacent to each other, or the inserted amino acids are not adjacent to each other, as long as the polypeptide has keratin characteristics and cell - proliferation - promoting activity.

[0044] The above - mentioned "deletion", that is, amino acid deletion treatment, means deleting 1, 2 or more than 3 amino acids in the sequence of any one of the 4 keratin amino acid sequences, as long as the polypeptide has keratin characteristics and cell - proliferation - promoting activity.

[0045] It should be noted that in the embodiments of the present application, the substitution of the recombinant keratin polypeptide may be a conservative amino acid substitution, which means that compared with the amino acid sequence of any one of the 4 keratin amino acid sequences, 3, more preferably 2 or 1 amino acid is replaced by an amino acid with similar or close properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 1.

[0046] The embodiments of the present application provide a polynucleotide, and the polynucleotide encodes the recombinant keratin polypeptide as described in the foregoing embodiments.

[0047] In an alternative embodiment, the nucleotide sequence of the polynucleotide is as 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.

[0048] An expression vector provided in an embodiment of the present application includes the polynucleotide as described in the foregoing embodiment. A host cell provided in an embodiment of the present application includes the expression vector as described in the foregoing embodiment. In an alternative embodiment, the host cell is Escherichia coli or yeast.

[0049] A method for preparing the recombinant keratin polypeptide as described in the foregoing embodiment provided in an embodiment of the present application includes:

[0050] Step S1, culturing the host cell in a production medium.

[0051] It should be noted that current research on keratin materials mainly focuses on the extraction and processing techniques of natural keratin. Traditional extraction methods mostly use high temperature, high pressure, or strong acid and strong base treatment. Such drastic chemical and physical treatment methods not only easily damage the natural structure of keratin but may also lead to changes in keratin network assembly and the generation of a large number of by-products. These by-products may, in some cases, affect the biological characteristics and performance of keratin, restricting its application in some sensitive fields (such as skin repair and regenerative medicine). To overcome these problems, some researchers have attempted to use genetic engineering techniques to produce keratin using transgenic cells or recombinant protein expression systems. The keratin materials obtained in this way can maintain relatively high stability in terms of structure and function.

[0052] However, existing genetic engineering techniques still face certain challenges, especially in the purification process of recombinant keratin. It has been found that by expressing the full-length sequence of keratin genes such as KRT81 or by performing amino acid substitution and modification on its sequence, although recombinant keratin can be effectively obtained, this recombinant protein still has impurity components with low molecular weights, such as substances like melanin and keratin-associated proteins (KAPs). These impurities not only increase the purification difficulty of recombinant keratin but may also have a negative impact on the biological activity of keratin and the final material properties. Existing purification techniques are still difficult to achieve the effect of efficiently removing these impurities, resulting in unsatisfactory purity and homogeneity.

[0053] In summary, there are various problems in the development and application of existing keratin materials, such as structural damage caused by extraction methods, difficulty in removing impurities during the purification process, and insufficient stability and functionality of recombinant proteins. These technical defects have restricted the in-depth application of keratin in a wider range of fields to a certain extent, especially in the field of high-end biomedical materials that require high purity and functionality of keratin.

[0054] Therefore, in the embodiments of the present application, a method for preparing recombinant keratin polypeptide is provided to specifically solve the above defects.

[0055] As described above, the host cell can be the host cell described in the foregoing embodiment.

[0056] This step may specifically include:

[0057] (1) Construction of Escherichia coli and yeast genetically engineered bacteria.

[0058] (2) Fermentation culture of Escherichia coli and yeast genetically engineered bacteria to obtain host cells.

[0059] In the above steps, it may involve selecting host cells and preparing a culture medium, then establishing culture conditions and performing culture. Inoculate the host cells into a culture medium containing the corresponding antibiotic, perform shake flask culture to obtain a host cell bacterial solution, inoculate the bacterial solution and the culture medium together into a culture medium containing the corresponding antibiotic, continue shake flask culture, and then add an inducer for induced expression.

[0060] Step S2: Isolate the recombinant keratin polypeptide from the host cell.

[0061] For example, in the above step S2, it may specifically include:

[0062] (3) Induce and express the recombinant keratin polypeptide for the obtained host cells;

[0063] (4) Purification of the recombinant keratin polypeptide.

[0064] After fermentation is completed, collect the bacterial cells by centrifugation or a tangential flow filtration system. The centrifugation method is fast and efficient and is suitable for large-scale production. The collected bacterial cells need to release the recombinant keratin polypeptide through a lysis method. Lysis methods include ultrasound, homogenization, high pressure, hypotonicity, lytic enzymes, organic solvents, etc. During the lysis process, conditions such as temperature, pH value, time, and pressure need to be controlled to avoid protein inactivation or degradation. After lysis is completed, use a centrifugation device in combination with a tangential flow microfiltration system to clarify the solid-liquid separation of the feed liquid and collect the supernatant.

[0065] During the above purification process, the following purification methods may be included: Preliminary purification: The recombinant keratin polypeptide is separated and preliminarily purified by multi-step chromatography (such as affinity chromatography, ion exchange chromatography, size exclusion chromatography). High-pressure chromatography purification: The recombinant keratin polypeptide is further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain a target protein with high purity. Post-purification treatment: The purified recombinant keratin polypeptide solution can be lyophilized to obtain a powdery product.

[0066] Through the above detailed steps, the efficient production, separation, and purification of the recombinant keratin polypeptide can be achieved. These technical details and optimization steps help to improve the yield and purity of the recombinant keratin polypeptide, meeting the requirements in fields such as biomedicine.

[0067] In an embodiment of the present application, a composition is provided, including the recombinant keratin polypeptide as described in the foregoing embodiment.

[0068] In an embodiment of the present application, a pharmaceutical product is provided, which contains the composition as described in the foregoing embodiment;

[0069] In an alternative embodiment, the pharmaceutical product is a pharmaceutical composition, a medical device, or a tissue engineering product;

[0070] In an alternative embodiment, the pharmaceutical composition is a topical preparation;

[0071] In an alternative embodiment, the topical preparation includes a topical microneedle preparation, a topical hydrogel preparation, or a topical infiltration preparation.

[0072] In an embodiment of the present application, an application of the recombinant keratin polypeptide as described in the foregoing embodiment in the preparation of medical device supplies or tissue engineering products is provided.

[0073] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0074] The test materials and reagents used in the examples are as follows:

[0075] 1. Strains, cells, and vectors: Strains containing the target gene and expression plasmids, such as Escherichia coli DH5a, TOP10, DE3, Rosetta(DE3), BL21(DE3) PLysE, BL21(DE3) PLysS, etc., were purchased from Beyotime, and Pichia pastoris X33 (Miaoling Biology). The vectors pet28a, vector pPICzalpha, and the antibiotic Zeocin were purchased from Invitrogen.

[0076] 2. Kits and enzymes: LDH detection kit (Roche 04744926001), improved Bradford protein assay kit (Sangon Biotech), seamless cloning kit In-Fusion Snap Assembly Master Mix (TaKaRa), plasmid extraction kit (General Biosystems), universal gel extraction kit (General Biosystems), restriction endonucleases, Q5 ultra-fidelity 2x Master Mix PCR polymerase, etc. were purchased from NEB.

[0077] 3. Media: The 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 supplemented with ampicillin at a final concentration of 100 μg / mL, LB + Kana medium is LB medium supplemented with kanamycin at a final concentration of 25 μg / mL, and LB + Zeo is LB medium supplemented with 100 μg / mL of Zeocin.

[0078] The induction medium for Escherichia coli is TB medium (2% yeast powder, 1% peptone, 1.5% dipotassium hydrogen phosphate, 2% potassium dihydrogen phosphate, adjusted to pH 7.0).

[0079] The high-density fermentation medium for Escherichia coli is TB medium supplemented with 0.1% - 0.2% antifoaming agent, with a pH of 7.0, and fed-batch glycerol at 400 g / L.

[0080] The yeast media are 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 supplemented with 100 μg / mL of Zeocin.

[0081] YPD medium is YPD medium supplemented with 100 μg / mL of Zeocin.

[0082] The yeast induction media are 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)).

[0083] The recombinant yeast fermentation medium is BSM medium (0.5% potassium dihydrogen phosphate, 5% diammonium hydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 0.1% calcium sulfate, 2% potassium sulfate, 0.03% antifoaming agent). After autoclaving the BSM medium, 4.35 mL of trace salt solution PTM1 is added. The composition of PTM1 is as follows: 6.5% ferric sulfate heptahydrate, 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, 0.02% biotin.

[0084] 4. Protein purification materials: Ni-NTA filler and nickel column are both purchased from Sanji Biotechnology.

[0085] 5. Skin soothing experiment materials: The raw material of Deshumin (SymCalmin®, 143535) is purchased from Symrise.

[0086] Example 1: Gene synthesis of recombinant keratin polypeptide

[0087] Experimental method:

[0088] (1) Gene analysis: The amino acid sequence of natural human hair keratin KRT81 (Genebank: AAH06452.1) is selected for physicochemical property analysis such as hydrophilicity-hydrophobicity and isoelectric point. The helical regions Coil1, Coil2 and hydrophilic conserved regions in human keratin KRT81 are preferably recombined, as Figure 1 shown.

[0089] The recombinant sequences are codon-optimized for Escherichia coli and Pichia pastoris codons. The newly obtained genes by recombination are the genes KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7 and KRT81 in this example, and the amino acid sequences are as SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, and SEQ ID NO.13.

[0090] (2) Gene synthesis: The full lengths of the genes KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7 and KRT81 are 687 bp, 735 bp, 647 bp, 450 bp and 1518 bp respectively. The sequences after codon optimization are as follows:

[0091] 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.

[0092] The optimized sequences were subjected to gene synthesis. The gene fragments were provided by General Gene Biotechnology Co., Ltd. The synthesized gene fragments of KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7, and KRT81 were inserted into the expression vectors of PET28a or PET24a using BamHI and HindIII, NcoI and XhoI respectively. Then the vectors were transformed into Escherichia coli TOP10 or DH5α for storage.

[0093] The vector maps constructed using the KRTBERI4 gene as a representative are shown respectively in Figure 2 .

[0094] Example 2: Construction and Induced Expression of Recombinant Keratin Polypeptides

[0095] Experimental Method:

[0096] 1. Construction of Escherichia coli Genetic Engineering Strains:

[0097] (1) Plasmid Extraction: Referring to Example 1, different strains containing the target gene were streaked on LB+A or LB+K plates containing the corresponding resistance and cultured overnight at 37°C. Subsequently, single colonies were selected from the overnight cultured plates and inoculated into 10 mL of LB+A or LB+K liquid medium, and continued to be cultured overnight at 37°C. An appropriate amount of the overnight cultured bacterial solution was centrifuged at 5000 rpm for 5 min, and the supernatant was removed to collect the bacterial cell pellet. The plasmid was extracted using the Tiangen Plasmid Extraction Kit. The concentration of the extracted plasmid was measured by NanoDrop and directly used for transformation of the expression host or stored at -20°C as needed.

[0098] (2) Strain Construction: Take 2 μL of the plasmid containing the target gene and place it in 100 μL of Escherichia coli competent cells, and let it stand on ice for 15 - 30 min. Heat shock this mixture in a 42°C water bath for 45 s, let it stand on ice for 2 - 3 min, add 500 μL of LB liquid medium without resistance, and culture it at 37°C and 190 rpm for 20 min. Take 100 μL of this bacterial solution and spread it evenly on LB+K or LB+Amp plates. Culture it overnight at 37°C until clear visible colonies grow.

[0099] (3)Induced expression of target protein: Pick monoclonal colonies from the overnight culture plate and culture them overnight in 10 mL of LB+A or LB+K liquid medium. Then transfer them to TB medium for scale-up culture at a ratio of 1%. When the OD600 of the bacterial solution reaches 0.5 to 0.8 at 37°C and 190 rpm, add IPTG with a final concentration of 0.1 to 0.5 mM for induced expression. The induction conditions are 16 - 26°C and 190 rpm for 16 - 20 h. Centrifuge to collect the bacteria, wash them repeatedly 3 times with phosphate buffer, and the washed bacteria can be stored at -20°C or directly lysed with a high-pressure cell disruptor and then centrifuged at 10000 rpm for 30 min. Take the supernatant after centrifugation and store it.

[0100] (4)SDS-PAGE detection of target protein: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, place it in boiling water at 100°C for 10 min, and then add 20 μL to each well of the SDS-PAGE protein gel. Run at 80 V for 1 h first, and then switch to 120 V until the bands are completely separated. Stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 30 min, and then decolorize it overnight with protein decolorizing solution (10% acetic acid, 5% ethanol).

[0101] The results are shown in Figure 3 , and the electrophoretic detection molecular weights of the proteins of genes such as KRTBERI4, KRTBERI5, KRTBERI6, KRTBERI7, and KRT81 are approximately 30 kDa, 30 kDa, 32 kDa, 20 kDa, and 50 kDa respectively.

[0102] 2. Construction of yeast genetic engineering bacteria:

[0103] (1)Plasmid extraction: Streak the different strains containing the target gene in Example 1 on the corresponding resistant LB+Z plates and culture them overnight at 37°C. Pick the monoclonal colonies from the overnight culture and inoculate them into 10 mL of LB+Z liquid medium respectively, and culture them overnight at 37°C. Take the overnight cultured bacterial solution, centrifuge at 5000 rpm for 5 min, discard the supernatant and collect the bacteria. Extract the plasmid using the Tiangen Plasmid Extraction Kit. Determine the concentration of the extracted plasmid with NanoDrop and then store it at -20°C.

[0104] (2) Plasmid linearization and recovery: Take 20 μg of the extracted plasmid, and use PmeI to perform single enzyme digestion at 37°C for 3 - 6 h. Take 5 μL of the linearized gene fragment and detect it with 1% agarose gel to confirm that the fragment is completely linearized. Use the 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 it to the collection column balanced with BL. Centrifuge at 12000 rpm for 1 min, wash it twice with PW solution, and centrifuge at 12000 rpm for 2 min. Transfer the collection column to a clean 1.5 mL centrifuge tube, air dry it at room temperature for 5 min, add ddH2O preheated at 55°C, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain the linearized gene fragment.

[0105] (3) Strain construction: Take 10 μL of the linearized fragment and add 100 μL of Pichia pastoris competent cells. Mix well on ice and transfer it to the electrode cup. Perform electroporation at 150 V and 200 mA for 10 ms. Immediately add 500 μL of recovery medium after electroporation, mix well and transfer it to a 1.5 mL centrifuge tube, and let it recover statically in a 30°C incubator for 3 h. Take 100 μL and spread it on the YPG + Z resistant plate, and incubate it upside down in a 30°C incubator for 2 - 3 d.

[0106] (4) Induction culture: Add BMGY to a 24-well plate, pick the yeast on the well-cultured plate into the 24-well plate, shake and culture it in a 30°C shaker for 1 day, add 100 μL of BMMY medium for induction, and continuously induce and culture for two days. After the culture is completed, centrifuge at 4000 rpm for 5 min, take the supernatant for preservation or directly perform SDS-PAGE.

[0107] (5) Protein expression detection: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, place it in boiling water at 100°C for 10 min, and then add 20 μL to each well of the SDS-PAGE protein gel. First run at 80 V for 1 h, and then transfer to 120 V until the bands are completely separated. Stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 30 min, and then decolorize it overnight with protein decolorizing solution (10% acetic acid, 5% ethanol).

[0108] The results are shown in Figure 4 , and the molecular weight detected by protein electrophoresis of KRTBERI7 is about 17 kDa.

[0109] Example 3: Purification of recombinant keratin polypeptide

[0110] Experimental method:

[0111] (1)Crude protein treatment: Take the frozen bacteria after washing with phosphate buffer, weigh them, resuspend the bacteria with 8 times the volume of lysis buffer (50 mM PB, 10 mM imidazole, 0.5 M NaCl, pH 7.0), and use a high-pressure homogenizer to break them under low-temperature conditions. The broken mixture is centrifuged at 4°C, 10,000 rpm for 25 min, the precipitate is discarded, the supernatant is harvested, and the volume is measured.

[0112] (2)Purification of protein by Ni-NTA nickel column: Rinse with (50 mM PB, 0.5 M NaCl, pH 7.0) for 5 column volumes to balance the nickel column: Take the pretreated protein for loading. Rinse with a 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.

[0113] Collect protein samples eluted with different concentrations of imidazole, and detect the purity by SDS-PAGE. After collection, rinse the nickel column with 20% ethanol, and store it after filling with ethanol.

[0114] For nickel columns reused multiple times, nickel column regeneration is required. The regeneration steps are as follows: Rinse with ddH2O for 5 column volumes, rinse with EDTA solution for 5 column volumes, rinse with NaOH solution for 5 column volumes, fill the affinity column with NiSO4 solution, and rinse for 5 column volumes.

[0115] Take different received protein purification solutions and perform SDS-PAGE detection to determine the purity and size of the target protein.

[0116] The results are shown in Figure 5 , and the molecular weight of KRTBERI7 detected by protein electrophoresis is about 20 kDa.

[0117] Example 4: Test on the cell proliferation-promoting activity of recombinant keratin

[0118] Experimental method:

[0119] (1)Culture of L929 cells: Place L929 cells in DMEM medium containing 10% fetal bovine serum and double antibodies, and culture them in a carbon dioxide incubator at a concentration of 5%, relative humidity of 95%, and a constant temperature of 37°C. Replace the medium every two days, and passage the cells after they are basically confluent on the fifth day.

[0120] First, aspirate the old medium, wash twice with PBS solution, and then add 0.5 mL of 0.25% trypsin for digestion. Observe under an inverted microscope. After the cells shrink and become round, add 10 mL of DMEM medium to stop digestion.

[0121] Gently pipette the cells to resuspend them, centrifuge at 1000 r / min for 5 min, discard the supernatant, add an appropriate amount of serum-containing medium, gently pipette again to make a cell suspension, and take 50 μL for counting.

[0122] For L929 cells in the logarithmic growth phase, aspirate the upper layer of the medium, wash twice with PBS solution, digest into single-cell suspension with 0.5 mL of 5% DMEM - 0.25% trypsin, wash twice again with PBS solution, and directly collect the cells growing in suspension.

[0123] (2)MTT assay: Select a 96-well plate, seed 2×10 4 cells per well, and set up experimental groups, blank control groups (containing only PBS solution), and calibration and zero-adjustment groups (without cells). Two replicate wells are set up for each group.

[0124] After 24 h, by microscopic observation, it is found that most cells have adhered and spread. At this time, remove the non-adherent cells and liquid in the wells. Adjust the polypeptide concentration in the experimental group to 0.5 mg / mL and add 20 μL, while the blank control group adds 20 μL of PBS buffer, and the calibration and zero-adjustment group only adds DMEM medium.

[0125] When adding samples, ensure that the pipette tip is close to the well wall and slowly inject. After adding each well, gently pipette to mix. Then let it stand for 30 min, and place the plate in a CO2 incubator. Observe the cell proliferation after culturing for 72 h. Use the MTT method to measure the absorbance (OD) value. Add 20 μL of MTT solution to each well of the 96-well plate and continue to culture for 4 h to complete the color reaction. Then terminate the culture, aspirate the culture medium in the wells or cover a layer of filter paper on the culture plate, quickly turn over the culture plate, add 150 μL of DMSO to each well, shake for 10 min to fully dissolve the formazan, and finally use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 490 nm.

[0126] Experimental results:

[0127] Table 1. Detection data of the cell proliferation-promoting activities of recombinant keratin and full-length KRT81-expressed protein in this example

[0128]

[0129] The results are shown in Figure 6As shown in Table 1, compared with the blank and the full-length expressed KRT81 protein, the recombinant keratins KRTBERI4, KRTBERI5, KRTBERI6, and KRTBERI7 have better effects in promoting the 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 for promoting cell proliferation.

[0130] Example 5: Test on the cell proliferation-promoting activity of recombinant keratin

[0131] In this example, to confirm whether the recombinant keratin polypeptide has the effect of skin soothing, a model of inflammatory edema stimulation of the skin was established using histamine. After modeling, different experimental samples were applied to test the human skin soothing effect of the recombinant keratin polypeptide. The specific steps are as follows:

[0132] (1) Inflammatory modeling: Before the experiment, the subjects need to clean the flexor sides of both forearms with dry paper towels, expose the flexor sides of the forearms, place them in a test state, keep relaxed, and avoid touching the test areas. The test areas are the flexor sides of both forearms, which are divided into four measurement areas A, B, C, and D, and the area of each area is 16 square centimeters (4 cm × 4 cm). Use an Antera 3D skin imaging measurement instrument to take pictures of the forearms and measure the skin redness values of each test area, and the measured values are used as the data before modeling. Apply a low-sensitivity medical tape to the middle of the test area within the designated area, press it, and then tear off the tape from one end of the skin and repeat 10 times. Then, apply a non-woven fabric of 3*3 cm completely soaked with a 1% histamine solution to the test areas of the subjects' both forearms for 10 min. Remove the non-woven fabric, and after the surface is dry without solution residue, use the Antera 3D skin imaging measurement instrument to take pictures and measure the skin redness values at the center positions of each test area, and the measured values are used as the T modeling data after modeling.

[0133] (2) Sample testing: After the T modeling data is collected, apply the samples to test the soothing effect. Set the blank (without any treatment), negative control (apply placebo after modeling), recombinant keratin (apply recombinant keratin after modeling), and positive control (apply Dermasooth after modeling) respectively. The subjects need to perform the same index detection 15 minutes, 1 hour, and 2 hours (T15min, T60min, and T120min) after using the products. If the change results of any parameter in the instrument test parameters at any visit before and after the test group (side) uses the product are significantly different (P < 0.05), or the test value results after using the sample are significantly better than those of the control group (side) (P < 0.05), then it is determined that the test product has the corresponding effect, otherwise it is considered that the test product has no corresponding effect.

[0134] The results are shown in Figure 7, compared with the control group, all 4 recombinant keratin polypeptides of the present invention have good soothing effects.

[0135] Example 6: Skin Capillary Regeneration Test

[0136] In this example, to study and confirm whether the recombinant keratin polypeptide has an effect on skin angiogenesis to improve local skin circulation, male mice were depilated and then topically applied with 5% recombinant keratin polypeptide solution and blank control (5% ethanol) respectively every day, with 8 mice in each group. After the experiment on the 15th day, the mice were sacrificed, skinned, and the skin of the test area on their backs was sectioned. The skin sections were immersed in physiological saline, and a Nikon SMZ18 stereomicroscope was used to assist in observing the skin blood vessels.

[0137] The results are as Figure 8 shown. Using the 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 this invention patent all have the function of promoting capillary neovascularization and improving local circulation.

[0138] 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 foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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 as 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, wherein The nucleotide sequence of the polynucleotide is shown as 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 according to claim 2.

5. A host cell, characterized in that, Comprising the expression vector according to claim 4.

6. The host cell according to claim 5, wherein, The host cell is Escherichia coli or yeast.

7. A method for preparing the recombinant keratin polypeptide according to claim 1, characterized in that, Comprising: Culturing the host cell according to claim 5 in a production medium; Isolating the recombinant keratin polypeptide from the host cell.

8. A composition, characterized in that, Comprising the recombinant keratin polypeptide according to claim 1.

9. A pharmaceutical product, characterized in that, The pharmaceutical product contains 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 or a tissue engineering product.

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

12. The pharmaceutical product according to claim 11, wherein, 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 device supplies and tissue engineering products.

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