A recombinant keratin injectable gel and preparation method thereof

Abstract

The present application relates to the field of medical supplies, and specifically provides a recombinant keratin injectable gel and a preparation method thereof. The injectable gel can form a recombinant keratin gel with reliable quality without adding a cross-linking agent while maintaining the mass ratio α of keratin and cysteine ​​at 5:1≤α<15:1 and adjusting to an alkaline pH value. The application of the injectable gel in medical products includes soft tissue filling, drug delivery, and cosmetic surgery. The injectable gel can also be used to promote fibroblast proliferation and migration, and promote fibroblast expression and secretion of collagen.

Description

Technical Field

[0001] The present application relates to the field of medical supplies, and in particular to a recombinant keratin injectable gel and a preparation method thereof. Background Art

[0002] The concept of soft tissue filling can be traced back to the 19th century, when autologous fat was first used for tissue repair. However, the survival rate of fat at that time was difficult to control, so permanent artificial synthetic fillers began to be used in the beauty industry. At present, the natural polymer materials used in the preparation of injectable gels in the medical and beauty industries are mainly animal collagen and sodium hyaluronate. Commonly used collagen comes from animal tissues such as pig skin, cowhide and bovine tendon. Animal collagen may have viral residues and cause immune rejection. Therefore, for some users, there are still rejection problems such as redness, swelling, allergies and chronic inflammation at the site of use. Although recombinant collagen solves the problem of viral origin, there are many types and it is difficult for consumers to distinguish them clearly. Since collagen gel cannot maintain its shape in the human body for a long time, it will be degraded into small molecule amino acids, peptides and other substances. Therefore, consumers will inject it multiple times, and the timeliness of use is relatively low. Unmodified hyaluronic acid hydrogel does not support integrin-mediated cell binding, and cell adhesive RGD peptide needs to be chemically coupled to the hyaluronic acid matrix. The whole process is cumbersome and not conducive to large-scale production. Keratin has a unique property and contains a large number of disulfide bonds, which has a good and stable water-locking effect. The self-crosslinking property can be used to obtain gel, which has a simple process and is stable in the body and not easy to degrade. It is an ideal filling material.

[0003] Keratin injection fillers have been used, but they are extracted keratin. The commonly used keratin is extracted from human hair, animal feathers, and animal scales by chemical methods. The molecular weight is not clear. It is a keratin mixture with problems such as immunogenicity and virus risks. Therefore, its development in the biomedical field is limited. Summary of the invention

[0004] The inventors of this application have overcome this defect in their previous research, and used recombinant keratin produced by genetic engineering technology to replace keratin extracted from animal sources. Recombinant keratin is based on the expression induced by recombinant plasmids, and high-purity recombinant keratin is obtained through fermentation, bacterial disruption, purification and other process steps.

[0005] This technology uses patented recombinant keratin as the main raw material, and through specific process steps, it is self-crosslinked to form a stable three-dimensional moisturizing gel. It does not need to add crosslinking agents such as glutaraldehyde to avoid the toxicity of residual crosslinking agents. The recombinant keratin used in this technology is derived from human hair genes, and has undergone certain design modifications to avoid immune problems. It is highly safe after being injected into the body and can be used as a filler for soft tissue filling, suitable for the field of medical beauty filling.

[0006] 1. An injectable gel, comprising:

[0007] Keratin and cysteine; the pH of the injectable gel is neutral.

[0008] 2. The injectable gel according to claim 1, wherein

[0009] The mass ratio α of keratin to cysteine ​​is 5:1≤α<15:1.

[0010] 3. The injectable gel according to claim 1, wherein

[0011] The keratin is prepared by mechanical method, acid-base treatment method, reduction method, oxidation method, electrochemical reduction method, copper-ammonia solution method, metal salt method, biological method or genetic engineering.

[0012] 4. Use of the gel described in any one of items 1 to 3 in the preparation of a filler for medical soft tissue repair or cosmetic use.

[0013] 5. A method for preparing an injectable gel, wherein:

[0014] Adding cysteine ​​to the above recombinant keratin stock solution, adjusting its pH to alkaline, and letting it stand for a period of time to form a gel;

[0015] Cut the gel into pieces and wash with plenty of purified water or PBS buffer until it becomes transparent;

[0016] The gel was broken up with a homogenizer, the precipitate was collected after centrifugation, and resuspended with physiological saline to form a stable recombinant keratin gel particle suspension.

[0017] 6. The method according to item 5, wherein the mass ratio of the added keratin stock solution to cysteine ​​is greater than or equal to 5:1 and less than 15:1.

[0018] 7. The method according to item 5, wherein the acid solution used to adjust the keratin solution after the addition of cysteine ​​is hydrochloric acid or citric acid.

[0019] 8. The gel according to item 5, wherein the pH adjustment to a weakly alkaline state refers to adjusting the pH of the keratin solution after the addition of cysteine ​​to a pH of 8.5 to 9.5.

[0020] 9. The method according to item 5, wherein the standing time is more than 3 hours.

[0021] 10. The method according to item 5, wherein the temperature of the cross-linking of the keratin solution is controlled to be maintained at 4-27°C.

[0022] 11. A filler comprising the gel according to any one of items 1 to 3 or the gel prepared by the method according to any one of items 5 to 10.

[0023] 12. The filler according to item 11, which is a filler for medical soft tissue repair or cosmetic use.

[0024] 13. Use of the gel described in any one of items 1 to 3 or the gel prepared by the method described in any one of items 5 to 10 for promoting the proliferation and migration of fibroblasts and promoting the expression and secretion of collagen by fibroblasts. This application has the following beneficial technical effects.

[0025] The present application provides the use of the above-mentioned injectable gel in medical products such as cosmetic injectable fillers and in the field of cosmetic plastic surgery, including soft tissue filling and drug delivery.

[0026] The recombinant keratin used in this application has the advantages of high activity, high purity, high safety and non-immunogenicity compared to ordinary keratin. There is no need to add various cross-linking agents during the gel production process, making it safer to use.

[0027] The covalent cross-linking of recombinant keratin and cysteine ​​absorbs a large amount of water. At the same time, the three-dimensional structure makes the gel have a very good water-locking ability. Finally, the recombinant keratin can be completely degraded into amino acids in the body. At the same time, based on the special properties of keratin, its degradation rate is also lower than that of traditional fillers. It can be used as a new type of human body filler and last longer.

[0028] The synergistic use of recombinant keratin and cysteine ​​has better antioxidant and collagen regeneration stimulating effects.

[0029] The cross-linking recombinant keratin method of the present application does not require the participation of a cross-linking agent, and cross-linking can be completed at room temperature to form a gel. The process is relatively simple, and the distribution of gel particle size is controllable, which can meet the needs of process transformation and mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a bar graph of the results in Experimental Example 4;

[0031] Figure 2 A. Figure 2 B is a comparison diagram of the gels formed in Example 1 and Comparative Example 6;

[0032] Figure 3 A is a photo of the rat 24 hours after the recombinant keratin gel was injected subcutaneously;

[0033] Figure 3 B is a photo of rats injected subcutaneously with recombinant collagen 24 hours later;

[0034] Figure 3C is a photo of a rat injected subcutaneously with normal saline only 24 hours later;

[0035] Figure 4 The results of H&E staining of rat skin tissue one week after the injection of recombinant keratin gel in Example 1;

[0036] Figure 5 A is the Masson staining of skin tissue sections of rats in the normal saline control group;

[0037] Figure 5 B is the Masson staining image of skin tissue sections of rats in the recombinant collagen group;

[0038] Figure 5 C is the Masson staining image of the skin tissue sections of rats in the recombinant keratin gel group. DETAILED DESCRIPTION

[0039] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the implementation of the present application, but the description is based on the general principles of the specification and is not used to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0040] The present application provides an injectable gel comprising keratin and cysteine.

[0041] In the present application, "keratin" is a fibrous natural protein, which is the main component of wool, human hair, hoof, horn, nail of mammals, and feathers of birds. Generally, according to the secondary structure classification, keratin can be divided into α-keratin (molecular weight of 40-60kDa) and β-keratin (molecular weight of 10-25kDa). The present application does not limit the type and source of keratin, and those skilled in the art can fully understand that the keratin can be of any natural source or synthesized by molecular biology.

[0042] Currently, the methods for preparing keratin that have been disclosed are mostly mechanical methods, acid-base treatment methods, reduction methods, oxidation methods, electrochemical reduction methods, copper ammonia solution methods, metal salt methods, and biological methods.

[0043] In the present application, mechanical extraction of keratin generally uses pressurization and heating to break the disulfide bonds between keratin molecules in the hair. However, mechanical extraction methods can usually only obtain proteins with lower molecular weights, or even polypeptide mixtures.

[0044] In this application specification, the oxidation method for extracting keratin is to use an oxidant to break the disulfide bonds in keratin and oxidize them into sulfonic acid groups to obtain keratin. The oxidant used is generally a peroxide such as performic acid, peracetic acid, and hydrogen peroxide. However, the phenomenon of peptide bond oxidation cleavage is easy to occur, and the average molecular weight of the obtained keratin is not high.

[0045] In the present application, the reduction method for extracting keratin is to use a reducing agent to reduce the disulfide bonds in keratin to thiol groups to obtain keratin. The reducing agent used is generally a thiol compound, such as sodium thioglycolate, thioglycolic acid, etc. However, the entire process is complicated, keratin is unstable, and the obtained solution will be re-oxidized and reduced to insoluble matter.

[0046] In this application specification, the acid-base treatment method for extracting keratin is to use strong acid and strong base reagents to swell the hair, and then hydrolyze it at a certain temperature to obtain soluble keratin, usually in combination with a reducing agent. However, the acid-base treatment process will inevitably cause hydrolysis of protein macromolecular peptide bonds and decomposition of disulfide bonds. The extraction process will produce alkaline wastewater and waste acid vapor, which will pollute the environment.

[0047] In this application, the biological method mainly uses keratinase produced by microorganisms to induce the decomposition of keratin, which is to enzymatically hydrolyze the higher structure of keratin into secondary structures such as amino acids and polypeptides. However, this method is generally time-consuming and the products are mostly polypeptides with low molecular weight.

[0048] In this application specification, in the process of preparing keratin by genetic engineering, genetically engineered bacteria are used to strengthen gene transcription and translation, achieve efficient expression and active secretion, and effectively improve the intensity of keratin production. Recombinant keratin is generally modified in performance, and the activity of extracellular protein is single, which simplifies the purification work downstream of fermentation. New keratin is sought through genetic engineering technology.

[0049] In the present application specification, cysteine ​​plays a vital role in the formation of protein gel. Protein gel is a three-dimensional structure formed by a cross-linked network between protein molecules, which is usually supported by disulfide cross-links between cysteine ​​residues. Cysteine ​​residues in proteins undergo oxidation reactions under acidic conditions to form disulfide bonds. These disulfide bonds connect different protein molecules together to form a stable cross-linked network structure. This network structure creates a mesh structure between protein molecules, thereby forming a three-dimensional structure of the gel. The number and position of cysteine ​​cross-links can regulate the properties of protein gels. By adjusting the reaction conditions, the content of cysteine ​​and the type of protein, the pore structure, pore size, pore distribution and cross-linking density of the gel can be controlled, thereby regulating the properties of the gel such as water absorption, stability and permeability. Cysteine ​​cross-links in protein gels not only affect the physical properties of the gel, but may also affect the biological activity and function of the protein. Cross-linking may lead to changes in protein conformation, thereby affecting its biological behaviors such as interaction, signal transduction and binding with other biomolecules in cells.

[0050] In this application specification, "disulfide bonds" are relatively stable chemical bonds, and their formation can increase the stability of the gel structure. Due to the presence of these cross-linking points, the protein molecules in the gel maintain a relatively fixed position relative to each other, thereby increasing the mechanical strength and durability of the gel.

[0051] In a specific embodiment of the present application, the keratin is keratin prepared by mechanical method, acid-base treatment method, reduction method, oxidation method, electrochemical reduction method, copper ammonia solution method, metal salt method, biological method or genetic engineering, keratin prepared by genetic engineering.

[0052] In a specific embodiment of the present application, the keratin is preferably recombinant keratin prepared by genetic engineering.

[0053] In a specific embodiment of the present application, the amino acid sequence of the keratin is as shown in SEQ ID NO.1 or is a conservative mutant of SEQ ID NO.1.

[0054] The present application provides a method for preparing the above-mentioned injectable gel, which comprises the following steps:

[0055] First, take an appropriate amount of recombinant keratin and dissolve it in physiological saline, adjust the pH to 11.0, and the proportion of recombinant keratin in the physiological saline is 5-15wt%, for example, it can be 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, 8wt%, 8.5wt%, 9wt%, 9.5wt%, 10wt%, 10.5wt%, 11wt%, 11.5wt%, 12wt%, 12.5wt%, 13wt%, 13.5wt%, 14wt%, 14.5wt%, 15wt%;

[0056] Add 1-2.5wt% cysteine ​​to the recombinant keratin physiological saline solution, for example, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt%; adjust the pH to 9.0±0.5 with hydrochloric acid, for example, pH=8.5, pH=8.6, pH=8.7, pH=8.8, pH=8.9pH=9.0, pH=9.1, pH=9.2, pH=9.3, pH=9.4, pH=9.5, and let it stand for about 3-5h to form a gel, for example, the standing time can be 3h, 3.5h, 4h, 4.5h, 5h;

[0057] Cut the gel into pieces and wash with purified water or PBS for 3-5 times until the gel is transparent. The volume ratio of the buffer solution to the gel is 20:1 to 50:1, for example, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1. At this time, the pH value of the gel drops to about 7.0.

[0058] The gel is broken up with a homogenizer, the precipitate is collected after centrifugation, and resuspended with physiological saline to form a stable recombinant keratin gel suspension, so that it can be injected into the body through a syringe.

[0059] In a specific embodiment of the present application, the temperature at which the recombinant keratin solution self-crosslinks to form a gel is 4-27°C, for example, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, or 27°C.

[0060] The present application provides the use of the above-mentioned injectable gel in medical products such as cosmetic injectable fillers and in the field of cosmetic plastic surgery, including soft tissue filling and drug delivery.

[0061] Experimental Example 1 Preparation of recombinant keratin

[0062] 1.1 Synthesis and screening of recombinant keratin amino acid sequences

[0063] The amino acid sequence of keratin is shown in SEQ ID NO.1. The protein has a total length of 416 amino acids. In its structural region, the domain formed by the first amino acid has strong hydrophobicity, and the active structure of keratin is mainly the middle α-helical structure. On this basis, the present application further modifies the amino acid sequence shown in SEQ ID NO.1: there are a large number of cysteine ​​residues at the front end of the original sequence, which is easy to form a large number of disulfide bonds and mismatches during the E. coli expression and purification process, resulting in reduced efficiency. After modification, the amino acid sequence of keratin of the present application is shown in SEQ ID NO.2, and the recombinant keratin of the present application also retains the active region of the amino acid sequence shown in SEQ ID NO.1. The modified keratin sequence is shown in SEQ ID NO.2 below, with amino acids 56-367 retained.

[0064] SEQ ID NO.1 is as follows:

[0065] MPYNFCLPSLSCRTSCSSRPCVPPSCHSCTLPGACNIPANVSNCNWFCEGSFNGSEKETMQFLNDRLASYLEKVRQLERDNAELENLIRERSQQQEPLLCPSYQSYFKTIEELQQKILCTKSENARLVVQIDNAKLAADDFRTKYQTELSLRQLVESDINGLRRILDELTLCKSDLEAQVESLKEELLCLKSNHEQEVNTLRCQLGDR LNVEVDAAPTVDLNRVLNETRSQYEALVETNRREVEQWFTTQTEELNKQVVSSSEQLQSYQAEIIELRRTVNALEIELQAQHNLRDSLENTLTESEARYSSQLSQVQSLITNVESQLAEIRSDLERQNQEYQVLLDVRARLECEINTYRSLLESEDCNLPSNPCATTNACSKPIGPCLSNPCTSCVPPAPCTPCAPRPRCGPCNSFVR

[0066] SEQ ID NO.2 is as follows:

[0067] KETMQFLNDRLASYLEKVRQLERDNAELENLIRERSQQQEPLLCPSYQSYFKTIEELQQKILCTKSENARLVVQIDNAKLAADDFRTKYQTELSLRQLVESDINGLRRILDELTLCKSDLEAQVESLKEELLCLKSNHEQEVNTLRCQLGDR LNVEVDAAPTVDLNRVLNETRSQYEALVETNRREVEQWFTTQTEELNKQVVSSSEQLQSYQAEIIELRRTVNALEIELQAQHNLRDSLENTLTESEARYSSQLSQVQSLITNVESQLAEIRSDLERQNQEYQVLLDVRARLECEINTYRSLL ESEDCNL

[0068] 1.2 Preparation of recombinant keratin

[0069] 1.2.1 Amplification of target fragment

[0070] 1) Synthesis of target gene

[0071] According to the codon usage preference of Escherichia coli in the Escherichia coli codon usage preference data table, the coding region sequence of the recombinant keratin protein that has been initially screened is optimized. On the premise of ensuring that the protein sequence of the recombinant keratin remains unchanged and only utilizing the degeneracy of the codons, the codons that are used less frequently in Escherichia coli and that may affect the efficiency of ribosome passage during the translation process are replaced with codons that are used more frequently to obtain a codon-optimized nucleic acid sequence. The obtained sequence is shown in SEQ ID NO.3 in the sequence listing.

[0072] According to the nucleic acid sequence of the target gene shown, a template gene was obtained through full gene synthesis and sequencing verification, as shown in SEQ ID NO: 3.

[0073] SEQ ID NO.3 is as follows:

[0074] GAAAAAGAAACCATGCAGTTTCTGAATGATCGTCTGGCGAGCTACCTGGAGAAAGTACGCCAGCTGGAACGCGATAATGCCGAACTGGAAAATCTGATTCGCGAACGCAGCCAGCAGCAGGAACCGCTGCTGTGCCCGAGCTACCAGAGCTATTTTAAAACCATTGAAGAACTGCAGCAGAAAATTCTGTGCACCAAAAGCGAAAACGCGCGCCTGGTTGTACAGATTGATAACGCCAAACTGGCGGCCGATGATTTCCGCACCAAATATCAGACCGAACTGAGCCTGCGCCAGCTGGTGGAAAGCGATATTAACGGTCTGCGCCGTATCCTGGATGAACTGACCCTGTGCAAATCCGATCTGGAAGCGCAGGTGGAAAGCCTGAAAGAAGAACTGCTGTGCCTGAAAAGCAACCATGAACAGGAAGTGAACACCCTGCGCTGCCAGCTGGGCGATCGTCTGAATGTGGAGGTGGATGCGGCCCCGACGGTGGATCTGAACCGCGTGCTGAACGAAACCCGTAGCCAATATGAAGCGCTGGTGGAAACCAACCGTCGTGAAGTGGAACAGTGGTTTACGACTCAGACCGAAGAACTGAATAAACAGGTGGTGAGTAGCTCAGAACAGCTGCAGTCATATCAGGCCGAAATCATTGAACTGCGCCGCACCGTGAACGCGCTGGAAATTGAACTGCAGGCCCAGCACAATCTGCGTGATAGCCTGGAAAATACCCTGACCGAAAGCGAAGCGCGCTATAGCAGCCAGCTGAGCCAGGTACAGAGCCTGATCACCAACGTGGAAAGCCAGCTGGCCGAAATTCGCAGCGATCTGGAACGCCAGAACCAGGAATATCAGGTGCTGCTGGATGTGCGCGCGCGCCTGGAATGCGAAATTAACACCTATCGCAGTCTGCTGGAAAGCGAAGACTGCAACCTG

[0075] 2) Design primers according to the nucleic acid sequence of the target gene. Use the synthesized template gene as a template and use F-KRT31:CCCATATGGAAAATCTGTATTTTCAGGGTGA (SEQ ID NO.4) and R-KRT31:CGGGATCCCAGGTTGCAGTCTTCGCTTTCCAG (SEQ ID NO.5) as primers for PCR amplification.

[0076] PCR reaction system: 1 μL of 10 μmol / L primer, 1 μL of target gene or linearized pET28a-His-TEV vector gene, 4 μL of dNTP (2.5 mM each), 10× Buffer (containing Mg 2+ )5μL, 1μL Pfu DNA Polymerase, and add water to make the total volume 50μL.

[0077] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 5 min.

[0078] 3) The PCR amplification product was detected by agarose gel electrophoresis. The amplified target fragment (about 1.0 kb) was the same size as the expected fragment, that is, the target gene fragment and the pET28a-His-TEV vector gene fragment were obtained.

[0079] 1.2.2 Construction of recombinant plasmid

[0080] 1) Add 4 μL of the purified target gene fragment, 6 μL of the pET28a-His-TEV linearized vector, 2 μL of 10XCloneEZBuffer, 2 μL of CloneEZ Enzyme, and deionized water to 20 μL, mix, keep at 22°C for 30 minutes, and then keep on ice for 5 minutes.

[0081] 2) Take 100 μL of DH5α or Top10 E. coli competent cells, add the above mixture, flick a few times, and incubate on ice for 30 minutes; heat shock in a 42°C water bath for 90 seconds, and incubate on ice for 5 minutes; add 1 mL of SOC medium to the cells, shake gently at 37°C for 1 hour at 200 rpm; centrifuge at 5000 rpm for 5 minutes to collect the cells, and resuspend the cells in 100 μL of SOC liquid medium.

[0082] 3) Spread the bacteria evenly on the antibiotic-containing plate and culture at 37°C overnight. Pick three positive clones and inoculate them in 5 mL LB medium containing 50 μg / mL kanamycin (Kan) antibiotics and culture them at 37°C, 220 rpm / min overnight.

[0083] 4) Take 3 mL of the bacterial solution of each sample to extract the plasmid (Tiangen Plasmid Extraction Kit), and send it to Nanjing GenScript for sequencing. The correctly sequenced plasmid is named pET28a-His-TEV-KERATIN.

[0084] 1.2.3 Construction of genetically engineered Escherichia coli

[0085] 1) Take BL21(DE3) competent cells and dissolve them in ice. Take 1μL of recombinant plasmid and add it to BL21(DE3) competent cells and mix gently. After standing in ice for 30 minutes, heat shock at 42℃ for 60 seconds and quickly return to ice. Then add 450μL of room temperature LB medium to the competent cells and shake at 37℃, 220rpm for 1 hour.

[0086] 2) Take 100 μL of bacterial solution from the tube and apply it to LB plate containing Kan antibiotics, and culture at 37℃ overnight. Pick 2 positive clones from the plate and inoculate them into 5mL LB medium containing 50μg / mL Kan antibiotics, and culture at 37℃, 220rpm for about 3h. When OD600=0.6, take the bacterial solution for seed preservation. That is, the genetically engineered E. coli bacteria are obtained.

[0087] 1.2.4 Inducing recombinant protein expression

[0088] 1) Take 20g of the collected precipitated bacterial cells and resuspend them in 100mL Tris buffer, which contains 50mMTris (pH 8.0), 500mM NaCl, and 5%wt Glycerol. Use a homogenizer to break them twice, mix them well, and centrifuge them at 4000rpm and 4℃ for 0.5h. Since the target protein exists in inclusion bodies, collect the precipitate for later use.

[0089] 2) The collected precipitate was resuspended and dissolved in 40 mL denaturing buffer (50 mM Tris (pH 8.0), 500 mM NaCl, 5% glycerol, 20 mM β-mercaptoethanol, 8 M urea, and the balance was deionized water). After full dissolution, centrifuge at 12000 rpm for 1 hour to take the supernatant, and then transfer the supernatant to a 5 mL HisFF affinity column (equilibrated with denaturing buffer).

[0090] 3) The column was rinsed with 10 times denaturation buffer. Then, the column was eluted in stages with denaturation buffers containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM and 500mM imidazole, and the eluted protein solution was collected and desalted with dialysate (25mMTris pH=8.0, 10mM imidazole, 20mMβ-mercaptoethanol, 20mM cysteine, and the remainder was deionized water) to exchange the buffer.

[0091] 4) Add TEV enzyme to the protein solution obtained in step 3), perform enzymatic hydrolysis at 25°C for 2 hours, remove the fusion HIS tag at the N-terminus of the protein, and collect the supernatant by centrifugation. Transfer to a 5 mL HisFF affinity column (balanced with dialysate), rinse with 3 times the dialysate, and collect the flow-through.

[0092] 5) Desalting by dialyzing with dialysate (20 mM β-mercaptoethanol, 20 mM cysteine, the remainder being deionized water) for 12 h, and freeze-drying to obtain the target protein.

[0093] Example 1 Preparation of recombinant keratin gel

[0094] Take the modified recombinant keratin freeze-dried powder of Experimental Example 1, add physiological saline and cysteine ​​successively, so that the final concentration of recombinant keratin is 10wt%, and the final concentration of cysteine ​​is 1wt%. The mass ratio of keratin to cysteine ​​is 10:1, and the pH value is adjusted to pH1=9.0 with 3M hydrochloric acid. The viscosity of the protein stock solution will change significantly. When the color of the solution changes from white transparent to milky white, stop stirring and let it stand at 25°C for about 4 hours to self-crosslink and form a gel. Cut the gel into pieces and wash it with PBS buffer until it becomes transparent. At this time, the pH value of the gel is pH2, which drops to about 7.0. After the gel is crushed with a homogenizer, the precipitate is collected and resuspended with physiological saline to obtain a stable recombinant keratin gel particle suspension with a gel particle size of 50-1000 microns, which can be directly extruded through an injection needle. This embodiment can produce a gel of reliable quality without adding a cross-linking agent, and the cross-linking effect and gel quality are the best in this embodiment.

[0095] Example 2

[0096] The difference compared with Example 1 is that the final concentration of the recombinant keratin is 5wt%. Since the content of the recombinant keratin is reduced, the cross-linking effect is slightly poor and the gel elastic modulus is low.

[0097] Example 3

[0098] The difference from Example 1 is that the gel is formed after standing for 3 hours. The cross-linking effect of this example is better and the gel quality is better.

[0099] Example 4

[0100] The difference from Example 1 is that the pH of the gel is adjusted to pH 1 = 9.0 with 5M hydrochloric acid and the gel is formed by standing for 2 hours. The cross-linking effect of this example is better and the gel quality is better.

[0101] Example 5

[0102] The difference from Example 1 is that the pH of the gel is adjusted to pH 1 = 9.0 with 1 M hydrochloric acid and the gel is formed by standing for 5 hours. This example has excellent cross-linking effect and good gel quality.

[0103] Example 6

[0104] The difference from Example 1 is that 3M citric acid is used to adjust the pH value. This example has excellent cross-linking effect and good gel quality.

[0105] Example 7

[0106] The difference from Example 1 is that the gel is formed by standing overnight at 4°C. Since the temperature is too low, the gel formation speed is too slow.

[0107] Example 8

[0108] The difference compared with Example 1 is that the final concentration of recombinant keratin is 15wt%, the final concentration of cysteine ​​is 1wt%, the mass ratio of keratin to cysteine ​​is 15:1, and the pH value is adjusted with hydrochloric acid. Since the concentration of recombinant keratin is too high, local severe aggregation is likely to occur when the pH value is adjusted, and it is difficult to effectively form a three-dimensional gel.

[0109] Comparative Example 1

[0110] The difference from Example 1 is that the recombinant keratin self-crosslinking stock solution was left to stand overnight at a temperature of 40° C., but due to the high temperature, it remained in a solution state and could not form a gel.

[0111] Comparative Example 2

[0112] The difference from Example 1 is that the pH of the gel was adjusted to pH 1 = 8.0 using 3M hydrochloric acid and allowed to stand for 5 hours. Since the pH was too low, no gel was formed.

[0113] Comparative Example 3

[0114] The difference from Example 1 is that the pH of the gel is adjusted to pH1=10.0 with 3M hydrochloric acid and the gel is left to stand for 4 hours to form. Since the pH is too high, the strength of the formed gel is low and it is easy to break.

[0115] Comparative Example 4

[0116] The difference from Example 1 is that the pH of the gel was adjusted to pH 1 = 9.0 with 3M hydrochloric acid and allowed to stand for 1 hour. However, since the standing time was too short, no gel was formed.

[0117] Comparative Example 5

[0118] The difference compared with Example 1 is that the final concentration of recombinant keratin is 15wt%, the final concentration of cysteine ​​is 0.8wt%, the mass ratio of keratin to cysteine ​​is 18.75:1, the pH is adjusted with hydrochloric acid, and no gel is formed after standing for more than 5 hours.

[0119] Comparative Example 6

[0120] The difference compared with Example 1 is that the final concentration of recombinant keratin is 15wt%, the final concentration of cysteine ​​is 0.5wt%, the mass ratio of keratin to cysteine ​​is 30:1, the pH is adjusted to pH1=9.0 with hydrochloric acid, and no gel is formed after standing overnight.

[0121] Table 1

[0122]

[0123]

[0124]

[0125] Note: The ratings of cross-linking degree from best to worst are “+++”, “++”, “+”, “-”, “--”; “None” means that gel cannot be produced under the current conditions.

[0126] Combined with the experimental data of each embodiment and comparative example shown in Table 1:

[0127] The effect of embodiment 1 is the best;

[0128] It can be seen from Example 2 that due to the reduced content of recombinant keratin, the cross-linking effect is slightly poor and the gel elastic modulus is low;

[0129] The cross-linking effects of Examples 3 to 6 are all excellent or relatively excellent, and the gel quality is good;

[0130] It can be seen from Example 7 that due to the low temperature, the gel formation speed is too slow, but the gel quality is good;

[0131] It can be seen from Example 8 that due to the high concentration of recombinant keratin, local severe aggregation is likely to occur when adjusting the pH value, and it is difficult to effectively form a three-dimensional gel;

[0132] It can be seen from Comparative Example 1 that due to the high temperature, the solution always remains and cannot form a gel;

[0133] It can be seen from Comparative Example 2 that gel cannot be formed due to too low pH;

[0134] It can be seen from Comparative Example 3 that due to the high pH, ​​the formed gel has low strength and is easily broken;

[0135] It can be seen from Comparative Example 4 that gel cannot be formed due to too short a standing time;

[0136] It can be seen from Comparative Examples 5-6 that gel cannot be formed due to the low cysteine ​​content.

[0137] The effect diagram of the recombinant keratin gel prepared by comparing Example 1 with Comparative Example 6 is as follows: Figure 2 A. Figure 2 As shown in B.

[0138] Experimental Example 2 Elastic modulus test

[0139] The injectable recombinant keratin gel prepared in Examples 1-7 was selected, and the elastic modulus of the injectable recombinant keratin gel was measured at a test temperature of 25° C., a slit width of 0.052 mm, and a vibration frequency in the range of 0.1-100 Hz. The elastic modulus at a frequency of 0.1 Hz was recorded, and the results are shown in Table 2.

[0140] Experimental Example 3 In vitro degradation study

[0141] Accurately weigh 0.5 g of the recombinant keratin gel particle suspension of Example 1-7, add 2 mL of phosphate buffer (0.1 mol / L, pH 7.0) and 2 mL of trypsin solution (0.1 g / mL, Biyuntian, batch number: 20190201), mix well, place in a 42 ° C water bath, take 50 μL of the mixed solution at different enzymatic hydrolysis time points and dilute to 3 mL, and use an ultraviolet spectrophotometer to measure the absorbance at 280 nm. The time when the absorbance no longer changes is the enzymatic hydrolysis time. The results are shown in Table 2.

[0142] Table 2

[0143] Group Elastic modulus(Pa) Degradation cycle (hours) Example 1 1429 27 Example 2 680 17.5 Example 3 963 20 Example 4 1107 26 Example 5 1080 26.5 Example 6 925 25 Example 7 1074 24

[0144] The results show that Examples 1-7 can produce gels of reliable quality without adding a crosslinking agent, among which the recombinant keratin gel prepared in Example 1 has the best quality, the highest elastic modulus of 1429Pa, and the longest degradation period of 27h. Based on the results of Examples 1-7, when the mass ratio α of recombinant keratin and cysteine ​​is within the range of 5:1≤α<15:1, the higher the concentration of recombinant keratin, the denser the gel network structure formed, the more stable the gel, and the longer the anti-enzymatic time. In addition, the longer the crosslinking time, the more complete the crosslinking of the recombinant keratin itself and the crosslinking with cysteine, and the better the stability of the formed gel.

[0145] Experimental Example 4 Cellular Activity Test of Recombinant Keratin Injection Gel

[0146] L929 cells were added to MEM culture medium (10% horse serum, 1% double antibody) and cultured in an incubator. The recombinant keratin gel in Example 1 was used to test the cell activity. The positive control group used a recombinant collagen product purchased on the market, and the blank control group used a prepared fresh culture medium. Then the recombinant keratin gel, recombinant keratin product and fresh culture medium were placed in a 96-well plate, and L929 cells were seeded in a 96-well plate at a density of 10,000 cells / well. The cells were cultured in an incubator at 5% CO2 and 37°C for 24 hours. After aspirating the supernatant, the different test groups prepared above were added respectively. After incubation for 24, 48 and 72 hours, CCK8 was added to the well plate. After 1 hour, the absorbance of the solution at 450nm was measured with an enzyme reader. The experimental results are as follows. Figure 1 As shown,

[0147] The results showed that recombinant keratin gel can promote the proliferation and differentiation of L929 cells and enhance cell activity. The effect of recombinant keratin gel on cell activity at 24h, 48h, and 72h was greater than that of the other two groups, and the effect of promoting cell proliferation was significantly better than recombinant collagen purchased on the market.

[0148] Experimental Example 5 Inflammatory Response Test of Recombinant Keratin Gel

[0149] 20 healthy adult mice were randomly divided into 4 groups, 5 mice in a group, and the back hair was removed and the skin was prepared with a depilatory agent. After cleaning the skin with water, the prepared skin of the mice was disinfected with iodine and deiodinated with 75% alcohol. Group 1 was a blank control without injection of any reagent, Group 2 was a negative control injected with normal saline, Group 3 was a positive control injected with commercially purchased recombinant collagen, and Group 4 was an experimental group injected with the recombinant keratin gel prepared in Example 1. Each mouse was injected subcutaneously with 0.2 mL, and the injected mice were placed in the same environment for feeding. After 3 days, 1 week, 2 weeks, 3 weeks and 5 weeks of feeding, one mouse was killed each time. The killing was performed by cervical dislocation, and then the skin at the injection site on the back was taken. The skin at the site of injection of the experimental material was cut off with a scalpel and fixed in neutral formaldehyde, and then stained with hematoxylin-eosin staining and photographed with an ordinary optical microscope for pathological tissue analysis to observe the inflammatory response. The experimental results are as follows Figures 3 to 5 shown.

[0150] The experimental results are as follows Figure 3 A. Figure 3 B. Figure 3 C shows that one week after injection, H&E examination showed no granulocyte infiltration and cell necrosis in the recombinant keratin gel group, recombinant collagen group and normal saline control group. The epidermis of the skin tissue recovered completely, and no obvious abnormalities were found in the dermis, skin appendage hair follicles, sebaceous glands and subcutaneous tissue (such as Figure 4As shown). After 1 week, 2 weeks, 3 weeks and 5 weeks of subcutaneous injection, the mice had no obvious inflammatory reaction. This shows that the recombinant keratin injectable hydrogel prepared in this application is a safer, non-toxic and biocompatible biomaterial, which can effectively solve the problems of poor biocompatibility and virus risks of traditional biomaterials. Figure 5 A. Figure 5 B. Figure 5 The Masson staining results shown in C indicate that after the injection of recombinant keratin gel, large areas of collagen fibers can be observed in the dermis, which are significantly higher than those in the recombinant collagen and saline control groups. This indicates that after the injection of recombinant keratin, it can stimulate the dermis fibroblasts to express and secrete collagen to a certain extent, achieving a better and more lasting filling effect.

Claims

1. A method for preparing an injectable gel, wherein: Take the recombinant keratin freeze-dried powder, add physiological saline and cysteine ​​successively, so that the final concentration of recombinant keratin is 10wt%, the final concentration of cysteine ​​is 1wt%, and the mass ratio of keratin to cysteine ​​is 10:

1. Use 3M hydrochloric acid to adjust its pH value to pH1=9.0, the viscosity of the protein stock solution will change significantly, and when the color of the solution changes from white transparent to milky white, stop stirring, and let it stand at 25°C for 4 hours to self-crosslink to form a gel; cut the gel into pieces, wash it with PBS buffer until it becomes transparent, and the pH value of the gel at this time is pH2=7.0; crush the gel with a homogenizer, collect the precipitate, and resuspend it with physiological saline to obtain a stable recombinant keratin gel particle suspension, the gel particle size is 50-1000 microns, and it can be directly extruded through an injection needle; The amino acid sequence of the recombinant keratin is shown in SEQ ID NO.

2.

2. A filler comprising the gel prepared by the method according to claim 1.

3. The filler according to claim 2, which is a filler for medical soft tissue repair or cosmetic use.

4. Use of the gel prepared by the method according to claim 1 in preparing products for promoting the proliferation and migration of fibroblasts, and promoting the expression and secretion of collagen by fibroblasts.

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