Recombinant III-type human collagen hydrogel as well as preparation method and application thereof

Abstract

The invention discloses recombinant III type human collagen hydrogel which comprises recombinant III type human collagen, an osmotic pressure regulator, water and an optional buffering agent, the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, butanediol, pentanediol, mannitol and sucrose; the amino acid sequence of the recombinant III-type human collagen is as shown in SEQ ID NO: 2, the proline hydroxylation degree of the recombinant III-type human collagen is 15.0%-17.5%, and the recombinant III-type human collagen has a complete triple helix structure. The recombinant III-type human collagen is not added with a chemical cross-linking agent or an enzymatic promoter for cross-linking, and the used reagents are free of cytotoxicity, sensitization and any safety risk. Therefore, the recombinant III-type human collagen hydrogel not only has no crosslinking agent residue and can form a three-dimensional network structure, but also has good biocompatibility and degradability, especially can improve the cell adhesion degree and promote the cell proliferation and migration ability, and provides a new choice for biomedical materials.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a recombinant type III human collagen hydrogel, a preparation method thereof and uses thereof. Background Art

[0002] Collagen is the most abundant, widely distributed and complex functional macromolecular protein in the human body, accounting for 25% to 30% of the total protein in the human body. It is not only an important component of the extracellular matrix (ECM), but also an important structural protein of connective tissue, playing the role of supporting organs and protecting the body in the human body. At present, at least 28 types of natural collagen have been discovered, which are widely present in various tissues and organs in humans and animals, especially in bones, tendons, skin, ligaments, cartilage, cornea and blood vessels. The most abundant collagen in the body is type I, type II and type III, accounting for about 80% to 90% of the total collagen in the body. They are all classic fiber-forming collagens, which form characteristic collagen fibers of many tissues.

[0003] Collagen is mainly synthesized by fibroblasts or cells of similar origin (such as osteoblasts, chondroblasts, epithelial or endothelial cells, etc.). The synthesis, processing and formation of collagen fibers involve a series of complex processes, including transcription, translation, post-translational modification, removal of terminal peptides, self-assembly of collagen molecules, and polymerization of collagen microfibrils. First, procollagen polypeptide chains (protocallagen) are synthesized in cells. Three polypeptide chains are twisted together to form procollagen (procollagen), which is then moved to the extracellular space. Under the action of procollagen N protease and procollagen C protease, the N-terminal peptide and C-terminal peptide of procollagen are removed respectively to form tropocollagen molecules (tropocollagen, de-terminated collagen). Procollagen molecules self-assemble to form collagen microfibrils, which are further polymerized into collagen fibers and collagen bundles.

[0004] Collagen is rich in amino acids and imino acids such as glycine, proline, and hydroxyproline. It is either a homotrimer formed by three identical α chains (such as [α1]3), or a heterotrimer formed by three different α chains (such as [α1]2(α2) or α1α2α3). The trimer is in the shape of a long and thin rod and constitutes the basic structural unit of collagen fibers. Each α chain contains about 1,000 amino acids and has a left-handed helical configuration. Three left-handed helical chains are intertwined to form a right-handed helical structure. A single α chain has a repeated amino acid sequence (Gly-XY). n, where X and Y are usually proline and hydroxyproline, respectively. Collagen has a triple helical structure that is unique to collagen. The hydroxyl group on hydroxyproline is crucial for the formation of intrachain and interchain hydrogen bonds, maintaining the stability of the triple helical structure of collagen, and forming stable collagen fibers to exert biological effects in vivo.

[0005] Collagen fibers in the body are formed by the self-assembly of collagen molecules with a triple helical structure. Adjacent molecules are intertwined to form stable intermolecular covalent crosslinks. The basic structural unit of collagen fibers is the tropocollagen molecule, which is about 1.5nm in diameter and 300nm in length. These basic structural units are connected head to tail and arranged in bundles to form collagen fibers.

[0006] The unique structural characteristics, physical and chemical properties, excellent biocompatibility and biodegradability of collagen make it an ideal biomedical material, playing an important role in dermal tissue filling, hemostatic dressings, artificial skin, orthopedic repair materials, and drug sustained-release materials. In particular, it has incomparable functions and effects in inducing its own collagen production, which are unmatched by other tissue filling materials. Many tissue fillers of non-human animal collagen have been launched on the market and injected into facial tissues to support and fill and correct wrinkles.

[0007] At present, the fillers or implants used in the field of soft tissue filling are mostly in the form of gels, and their materials include sodium hyaluronate, animal collagen, recombinant humanized collagen, etc. Although sodium hyaluronate gel has a good supporting effect, it lacks the function of inducing the production of its own collagen; animal collagen gel has a good supporting effect, but there are immune rejection reactions and virus risks caused by heterologous collagen; recombinant humanized collagen gel has differences in sequence structure composition and sequence length compared with natural human collagen, especially the difference in post-translational modification of natural human collagen, that is, it lacks supporting effect, does not have complete cell biological effects, and cannot induce the production of its own collagen. Gels of different compositions have many disadvantages, which may cause the product to easily induce chronic inflammation, allergic swelling, and lack of necessary biological activity due to heterologous sources or structural differences from natural human collagen during use. In addition to factors of the material itself, there are also reasons such as residual exogenous substances. For example, in order to obtain cross-linked polymer fibers with certain mechanical strength and degradation stability, the fibers are usually prepared by adding chemical cross-linkers or enzymatic cross-linking. Although different methods for removing cross-linkers and promoting cross-linking enzymes are adopted in the preparation process, there are still chemical cross-linkers and promoting cross-linking enzymes, which may easily induce cytotoxicity of cross-linker residues and immune response of promoting cross-linking enzyme residues during use.

[0008] Hydrogel is a three-dimensional network biomaterial formed by cross-linking of hydrophilic polymers. Hydrophilic macromolecules form a hydrophilic network through moderate physical or chemical cross-linking, which can absorb and retain a large amount of water. Due to its good biocompatibility, it is widely used in the field of biomedical materials, such as hydrogel scaffolds made of natural polymer collagen, used as burn dressings and artificial skin, etc. With the development of tissue engineering, hydrogels are widely used in implantable medical materials, wound repair dressings and drug release carriers.

[0009] The materials for preparing hydrogels include natural polymer materials and synthetic polymer materials. The two materials have different characteristics and advantages and disadvantages. Natural polymer materials have good biocompatibility, while synthetic polymer materials have good mechanical properties. Natural polymer materials are a type of macromolecule extracted from organisms or directly obtained from the natural environment. They have good biocompatibility and degradability. The natural polymer materials for preparing hydrogels are mainly animal collagen and sodium hyaluronate. Animal collagen has been widely used in medicine, tissue engineering, medical devices, cosmetics and other fields due to its good biocompatibility, biodegradability and in vivo biological activity. Usually, animal collagen comes from pig skin, cow skin, bovine Achilles tendon, fish skin, etc. Animal collagen has inevitable defects and deficiencies. In the application of implantable medical materials, the heterogeneity of animal collagen is prone to cause adverse reactions such as allergies, or immune rejection reactions caused by virus residues and heterologous host protein residues. Therefore, for some users, there are a series of problems such as allergies, redness, swelling or chronic inflammation at the site of use.

[0010] The use of genetic recombination to prepare collagen can effectively solve the problem of viral hazards of animal-derived collagen. Currently, the most reported method is to use Escherichia coli or yeast fermentation to express and prepare recombinant collagen. Due to the limitations of preparation technology, most of the collagen prepared by recombinant methods belongs to recombinant humanized collagen or recombinant collagen, which are parts or fragments of natural human collagen and parts or fragments of natural human collagen combined with other non-human collagen sequences. Due to the differences in structural sequence composition and length with natural human collagen, especially the lack of post-translational modification of collagen, it does not have the hydroxyproline level and triple helix structure that are unique to collagen in the body to play its biological role, resulting in no or only partial biological effect, which greatly limits its application as a biomedical material.

[0011] At present, there is no hydrogel prepared from recombinant full-length type III human collagen with high purity, proline hydroxylation degree similar to that of natural human collagen, complete triple helix structure, and biological effects consistent with those of natural human collagen, and its application as a biomedical material in tissue filling. Therefore, it is urgent to develop a recombinant type III human collagen hydrogel with high purity and biological effects consistent with those of natural human collagen. Summary of the invention

[0012] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a recombinant type III human collagen hydrogel and a preparation method thereof and uses thereof.

[0013] Based on this, in the first aspect of the present invention, the present invention provides a recombinant type III human collagen hydrogel. According to an embodiment of the present invention, the recombinant type III human collagen hydrogel includes recombinant type III human collagen, an osmotic pressure regulator, water, and optionally a buffer; the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, butanediol, pentanediol, mannitol and sucrose. In the present invention, the raw materials of the recombinant type III human collagen hydrogel (hereinafter referred to as hydrogel) are cross-linked without adding chemical cross-linking agents or enzymatic agents, and the reagents used are non-cytotoxic, non-allergenic and other safety risks. Therefore, the recombinant type III human collagen hydrogel of the present invention not only has no cross-linking agent residue and can form a three-dimensional network structure, but also has good biocompatibility and degradability, especially can improve cell adhesion, and promote cell proliferation and migration ability, for biomedical materials Provide a new choice.

[0014] In the second aspect of the present invention, the present invention proposes a method for preparing the recombinant type III human collagen hydrogel described in the first aspect. According to an embodiment of the present invention, the method comprises: subjecting the recombinant type III human collagen, an osmotic pressure regulator, water, and optionally a buffer to a first mixing treatment to obtain a recombinant type III human collagen hydrogel. The preparation method of the recombinant type III human collagen hydrogel in the present invention is simple, and no chemical cross-linking agent or enzymatic agent is required for cross-linking during the preparation process. The reagents used are non-cytotoxic, non-allergenic, and any other safety risks. The prepared recombinant type III human collagen hydrogel product is safe, free of viral hazards, and will not cause allogeneic rejection reactions. In particular, it is not only free of cross-linking agent residues, but also has good biocompatibility and degradability, and can especially improve cell adhesion and promote cell proliferation and migration.

[0015] In the third aspect of the present invention, the present invention provides a biomedical material. According to an embodiment of the present invention, the biomedical material comprises: the recombinant type III human collagen hydrogel described in the first aspect, or the recombinant type III human collagen hydrogel prepared according to the method described in the second aspect.

[0016] In the fourth aspect of the present invention, the present invention proposes the use of the recombinant type III human collagen hydrogel described in the first aspect, or the recombinant type III human collagen hydrogel prepared according to the method described in the second aspect, in the preparation of biomedical materials.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 The SDS-PAGE diagram of the recombinant type III human collagen sample from which the N-terminal telopeptide and the C-terminal telopeptide are removed in Example 1 of the present invention is shown;

[0020] Figure 2 The HPLC chart of the recombinant type III human collagen sample from which the N-terminal telopeptide and the C-terminal telopeptide are removed in Example 1 of the present invention is shown;

[0021] Figure 3 The figure shows the result of identifying the N / C terminus of recombinant type III human collagen using LC / MS technology in Example 2 of the present invention;

[0022] Figure 4 The L-hydroxyproline standard curve in Example 2 of the present invention is shown;

[0023] Figure 5 The figure shows the results of the near and far ultraviolet scanning analysis of the CD standard solution in Example 2 of the present invention;

[0024] Figure 6 The CD spectrum of the recombinant type III human collagen sample in Example 2 of the present invention is shown;

[0025] Figure 7 The results of the JHM09 recombinant type III human collagen cell migration test in Example 3 of the present invention are shown;

[0026] Figure 8 The vacuum drying scanning electron microscope (SEM) image of the JHM09 recombinant type III human collagen self-assembled fibers in Example 4 of the present invention is shown;

[0027] Fig. 9 The scanning electron microscope (SEM) image of the JHM09 recombinant type III human collagen self-assembled fiber ethanol dehydration in Example 5 of the present invention is shown;

[0028] Fig.10 A schematic diagram showing the results of the JHM09 recombinant type III human collagen hydrogel cell migration test in Example 6 of the present invention is shown. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0031] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0032] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0033] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0034] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0035] In this article, the term "biomedical materials" is defined as a class of natural or artificially synthesized special functional materials that are used to contact and interact with living systems and can diagnose, treat, replace, repair or induce regeneration of their cells, tissues and organs, also known as biomaterials.

[0036] In order to promote the high-quality development of new biomaterials and standardize the naming of recombinant collagen biomaterials, the National Medical Products Administration has issued the "Guidelines for the Naming of Recombinant Collagen Biomaterials", which clearly named "collagen with a full-length amino acid sequence encoded by a specific type of human collagen gene prepared by DNA recombinant technology and having a triple helix structure" as "recombinant human collagen"; "collagen with a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombinant technology, or a combination of functional fragments of human collagen" as "recombinant humanized collagen"; "collagen with a designed and modified specific gene encoded amino acid sequence or its fragment, or a combination of such functional amino acid sequence fragments, prepared by DNA recombinant technology, whose gene coding sequence or amino acid sequence has low homology with the gene coding sequence or amino acid sequence of human collagen" as "recombinant collagen". This shows that only recombinant human collagen is consistent with natural human collagen and has the same in vivo biological effects as natural human collagen.

[0037] Herein, recombinant human type III collagen is also referred to as recombinant human type III collagen stock solution or JHM09 stock solution; recombinant human type III collagen self-assembles to form recombinant human type III collagen self-assembled fibers, also referred to as JHM09 fibers or JHM09 collagen fibers.

[0038] Detailed description of the recombinant type III human collagen hydrogel of the present invention, its preparation method and its use

[0039] The present invention provides a recombinant type III human collagen hydrogel, a preparation method thereof and uses thereof, which will be described in detail below.

[0040] Recombinant human type III collagen hydrogel

[0041] In the first aspect of the present invention, the present invention provides a recombinant type III human collagen hydrogel. According to an embodiment of the present invention, the recombinant type III human collagen hydrogel includes recombinant type III human collagen, an osmotic pressure regulator, water, and optionally a buffer; the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, butanediol, pentanediol, mannitol and sucrose. In the present invention, the raw materials of the recombinant type III human collagen hydrogel (hereinafter referred to as hydrogel) are cross-linked without adding chemical cross-linking agents or enzymatic agents, and the reagents used are non-cytotoxic, non-allergenic and any other safety risks. Therefore, the recombinant type III human collagen hydrogel of the present invention not only has no cross-linking agent residue and can form a three-dimensional network structure, but also has good biocompatibility and degradability, especially can improve cell adhesion, and promote cell proliferation and migration ability, for biomedical materials Provide a new choice.

[0042] According to an embodiment of the present invention, the amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 2. The recombinant type III human collagen of the present invention is 100% homologous to the natural human collagen sequence, thereby further avoiding adverse reactions such as allergies caused by heterogeneity of the hydrogel and reducing immune rejection reactions.

[0043] According to an embodiment of the present invention, the purity of the recombinant type III human collagen is greater than 95%.

[0044] According to an embodiment of the present invention, the proline hydroxylation degree in the recombinant type III human collagen is greater than 15.0%, preferably 16.0% to 17.5%. The proline hydroxylation degree of the recombinant type III human collagen in the present invention is close to that of natural human collagen, thereby further reducing adverse reactions such as allergies caused by heterologous hydrogels and reducing immune rejection reactions.

[0045] According to a specific embodiment of the present invention, the degree of proline hydroxylation of the recombinant type III human collagen is 16.0%-17.5%. The hydroxyl groups on hydroxyproline are crucial for the formation of intra-chain and inter-chain hydrogen bonds, maintaining the stability of the triple helix structure of collagen, and forming stable collagen fibers to exert biological effects in vivo. When the degree of proline hydroxylation is 16.0%-17.5%, preferably 16.0%-16.9%, it is beneficial for the recombinant type III human collagen to form a triple helix structure and maintain the stability of the triple helix structure.

[0046] According to an embodiment of the present invention, the recombinant type III human collagen has a complete triple helix structure. The recombinant type III human collagen in the present invention has a complete triple helix structure, which is close to natural human collagen, thereby further avoiding adverse reactions such as allergies caused by heterogeneity of the hydrogel and reducing immune rejection reactions.

[0047] According to an embodiment of the present invention, in the circular dichroism spectrum, the recombinant type III human collagen has a negative absorption peak at a wavelength of 190 to 200 nm, and a positive absorption peak at a wavelength of 210 to 230 nm.

[0048] It should be noted that it is well known to those skilled in the art that the presence of both a negative absorption peak near 197 nm and a positive absorption peak near 221 nm in the circular dichroism spectrum is the gold standard for collagen to have a correct and complete triple helix structure.

[0049] According to an embodiment of the present invention, the recombinant type III human collagen has a negative absorption peak at a wavelength of 192-198 nm.

[0050] According to an embodiment of the present invention, the recombinant type III human collagen has a positive absorption peak at a wavelength of 215-225 nm.

[0051] According to an embodiment of the present invention, the circular dichroism spectrum of the recombinant type III human collagen is as follows: Figure 6 shown.

[0052] In an optional embodiment of the present invention, the recombinant type III human collagen consistent with natural type III human collagen in the present invention must have the following characteristics: (1) The structural composition is 100% homologous to the natural human collagen sequence, including being completely consistent with the full-length amino acid sequence composition and collagen length of natural human collagen; (2) The degree of post-translational modification, that is, the degree of proline hydroxylation is close to or consistent with that of natural human collagen, which is an important basis for the formation of the triple helix structure of collagen; (3) It has a complete triple helix structure, which is a necessary basis and condition for natural human collagen to exert its biological effects in vivo; (4) It has biological activity compared with animal-derived collagen; (5) The collagen fibers assembled from collagen have the typical characteristics of collagen fibers; (6) There must be no cross-linking agent residues in the assembled collagen fibers, as cross-linking agents are dangerous components that affect health.

[0053] According to an embodiment of the present invention, the recombinant human type III collagen is prepared by the following steps: constructing a genetically engineered bacterium expressing proline hydroxylase and full-length recombinant human type III collagen, wherein the amino acid sequence of the full-length recombinant human type III collagen is as shown in SEQ ID NO: 1; culturing the genetically engineered bacterium, and enzymatically digesting the culture product with pepsin to obtain the recombinant human type III collagen; wherein the copy number of the proline hydroxylase gene in the genetically engineered bacterium is between 3.00 and 4.00, and the ratio of the copy number of the proline hydroxylase gene to the copy number of the recombinant human type III collagen gene is (1.80 to 2.20):1.

[0054] The inventors of the present invention have found through experiments that the copy number of the proline hydroxylase gene and the ratio of the copy number of the proline hydroxylase gene to the copy number of the recombinant type III human collagen have a crucial influence on the degree of proline hydroxylation of the recombinant type III human collagen. When the copy number of the proline hydroxylase gene is too high or too low (not between 3.00-4.00), and the ratio of the copy number of the proline hydroxylase gene to the copy number of the recombinant type III human collagen gene is lower than 1.80:1 or higher than 2.20:1, the degree of proline hydroxylation of the recombinant type III human collagen will be insufficient, which will cause the recombinant type III human collagen to be unable to form a triple helix structure, or the formed triple helix structure has poor stability, resulting in a large difference from natural human collagen, causing adverse reactions such as allergies due to structural differences, leading to immune rejection reactions.

[0055] Therefore, the inventors of the present invention unexpectedly discovered that by regulating the copy number of the proline hydroxylase gene and the ratio of the copy number of the proline hydroxylase to the copy number of the recombinant type III human collagen, the degree of proline hydroxylation of the recombinant type III human collagen can be 16.0%-17.5%, which is beneficial for the recombinant type III human collagen to form a triple helix structure and maintain the stability of the triple helix structure. The hydrogel prepared using the above-mentioned recombinant type III human collagen can reduce adverse reactions such as allergies caused by structural differences and can reduce immune rejection reactions. According to an embodiment of the present invention, the ratio of the copy number of the proline hydroxylase to the copy number of the recombinant type III human collagen is (1.90-2.10):1.

[0056] In this article, unless otherwise specified, "full-length recombinant human type III collagen" refers to a protein without removing the N-terminal telopeptide and the C-terminal telopeptide, and its amino acid sequence is shown in SEQ ID NO: 1; "recombinant human type III collagen" refers to a protein without removing the N-terminal telopeptide and the C-terminal telopeptide, and its amino acid sequence is shown in SEQ ID NO: 2. In genetically engineered strains, the copy numbers of the full-length recombinant human type III collagen gene and the recombinant human type III collagen gene are the same.

[0057] In some embodiments of the present invention, the genetically engineered bacteria are obtained by the following steps: detecting the copy numbers of the P4H hydroxylase gene and the recombinant human type III collagen gene in the genetically engineered bacteria, calculating the ratio of the copy number of the P4H hydroxylase gene to the copy number of the recombinant human type III collagen gene, and screening genetically engineered bacteria in which the copy number of the proline hydroxylase gene is between 3.00 and 4.00 and the ratio of the copy number of the proline hydroxylase gene to the copy number of the recombinant human type III collagen gene is (1.80 to 2.20):1.

[0058] In one embodiment of the present invention, the detection of the proline hydroxylase gene copy number and the recombinant type III human collagen gene copy number are both performed by fluorescent quantitative PCR. Exemplarily, a double standard curve method can be used, and the internal reference gene is the Pichia pastoris housekeeping gene GAP (Glyceraldehyde-3-phosphate dehydrogenase) for detection.

[0059] According to an embodiment of the present invention, the recombinant human type III collagen exists in the form of recombinant human type III collagen self-assembled fibers.

[0060] According to an embodiment of the present invention, based on the total volume of the recombinant type III human collagen hydrogel, the weight volume percentage of the recombinant type III human collagen is 1.0% to 10.0%, for example, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0% and any two point values ​​therebetween as the range between the endpoint values, such as 3.0% to 10.0%, 3.5% to 10.0%, 4.0% to 10.0%, 4.5% to 10.0%, 5.0% to 10 .0%, 5.5%~10.0%, 6.0%~10.0%, 3.0%~9.0%, 3.5%~9.0%, 4.0%~9.0%, 4.5%~9.0%, 5.0%~9.0%, 5.5%~9.0%, 6.0%~9.0%, 3.0%~8.0%, 3.5%~8.0%, 4.0%~8.0%, 4.5%~8.0%, 5.0%~8.0%, 5.5%~8.0%, 6.0%~10.0%, 3.0%~7.0%, 3.5%~7.0%, 4.0%~7.0%, 4.5%~7.0%, 5.0%~7.0%, 5.5%~7.0%, 6.0%~7.0%. Thereby, the performance of the hydrogel can be further improved, and the cell adhesion, and the ability to promote cell proliferation and migration can be further improved.

[0061] In this article, without special explanation, the weight volume percentage of A refers to the percentage ratio of the weight of A to the volume of recombinant type III human collagen hydrogel (w / v, g / 100 mL).

[0062] According to an embodiment of the present invention, the recombinant type III human collagen self-assembly fiber is obtained by the following steps: the recombinant type III human collagen is subjected to a self-assembly treatment in a buffer solution. The recombinant type III human collagen self-assembly fiber is made of recombinant type III human collagen and does not contain a chemical cross-linking agent or an enzymatic agent. As a result, the recombinant type III human collagen self-assembly fiber has no cross-linking agent residue, can form a three-dimensional network structure, and has good biocompatibility and degradability.

[0063] According to an embodiment of the present invention, the buffer solution includes at least one of phosphate buffered saline, acetate buffered saline, citrate buffered saline, sulfate buffered saline, and Tris hydrochloride buffer;

[0064] According to an embodiment of the present invention, the pH value in the self-assembly process is 6.6-6.8.

[0065] According to an embodiment of the present invention, the self-assembly process is performed at room temperature.

[0066] According to an embodiment of the present invention, the temperature of the self-assembly treatment is 25°C to 35°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C and any two points therebetween as the range between endpoint values.

[0067] According to an embodiment of the present invention, the self-assembly process lasts for 20 hours to 24 hours.

[0068] According to an embodiment of the present invention, after the self-assembly process, the self-assembly product is subjected to a dehydration process.

[0069] According to an embodiment of the present invention, the dehydration treatment is performed by vacuum drying or ethanol dehydration.

[0070] According to an embodiment of the present invention, the weight ratio of the recombinant type III human collagen and the osmotic pressure regulator is (1.0-10): (0.1-1.0), for example, (3.0-10.0): (0.1-1.0), (3.0-10.0): (0.4-1.0), (3.0-10.0): (0.5-1.0), (3.0-10.0): (0.6- 1.0)、(3.0~10.0):(0.7~1.0)、(3.0~10.0):(0.8~1.0)、(3.0~9.0):(0.1~1.0)、(3.0~9.0):(0.4~1.0)、(3.0~9.0):(0.5~1.0)、(3.0~9.0):(0.6~1.0)、(3.0~9.0) :(0.7~1.0)、(3.0~9.0)、(0.8~1.0)、(3.0~8.0)、(0.1~1.0)、(3.0~8.0)、(0.4~1.0)、(3.0~8.0)、(0.5~1.0)、(3.0~8.0)、(0.6~1.0)、(3.0~8.0)、(0.7~1.0)、(3.0 ~8.0): (0.8~1.0), (3.0~7.0): (0.1~1.0), (3.0~7.0): (0.4~1.0), (3.0~7.0): (0.5~1.0), (3.0~7.0): (0.6~1.0), (3.0~7.0): (0.7~1.0), (3.0~7.0): (0.8~1.0). In this way, the performance of the hydrogel can be further improved, and the cell adhesion, and the ability to promote cell proliferation and migration can be further improved.

[0071] In an optional embodiment of the present invention, the weight ratio of the recombinant type III human collagen to the osmotic pressure regulator is (3.0-7.0): (0.4-1.0). This can further improve the performance of the hydrogel, further improve cell adhesion, and promote cell proliferation and migration.

[0072] According to an embodiment of the present invention, the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol and mannitol.

[0073] According to an embodiment of the present invention, the osmotic pressure regulator is sodium chloride.

[0074] According to an embodiment of the present invention, based on the total volume of the recombinant type III human collagen hydrogel, the weight volume percentage of the osmotic pressure regulator is 0.1% to 1.0%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% and any two points therebetween as the range between the endpoint values, such as 0.3% to 1.0%, 0.4% to 1.0%, 0.5% to 1.0%, 0.6% to 1.0%, 0.7% to 1.0%, 0.8% to 1.0%. Thus, the performance of the hydrogel can be further improved, and the cell adhesion and cell proliferation and migration abilities can be further improved.

[0075] According to an embodiment of the present invention, the weight ratio of the recombinant type III human collagen, the osmotic pressure regulator and the buffer is (1.0-10.0): (0.1-1.0): (0.5-5.0). This can further improve the performance of the hydrogel, further improve cell adhesion, and promote cell proliferation and migration.

[0076] In an optional embodiment of the present invention, the weight ratio of the recombinant type III human collagen, the osmotic pressure regulator and the buffer is (3.0-8.0): (0.4-1.0): (0.5-2.0). This can further improve the performance of the hydrogel, further improve the cell adhesion, and promote cell proliferation and migration.

[0077] According to an embodiment of the present invention, the buffer is selected from at least one of sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, potassium chloride, sodium acetate, potassium acetate, citric acid, sulfate and Tris hydrochloride.

[0078] According to an embodiment of the present invention, the buffer is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate and citric acid.

[0079] According to an embodiment of the present invention, the buffer is disodium hydrogen phosphate.

[0080] According to an embodiment of the present invention, based on the total volume of the recombinant type III human collagen hydrogel, the weight volume percentage of the buffer is 0.1% to 5.0%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% and any two point values ​​therebetween as the range between the endpoint values, such as 0.5% to 5.0%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2%, 1% to 5%, 1% to 4%, 1% to 3%, 1% to 2%. Thus, the performance of the hydrogel can be further improved, and the cell adhesion and cell proliferation and migration abilities can be further improved.

[0081] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 1.0 to 10.0 parts by weight of recombinant type III human collagen; and 0.1 to 1.0 parts by weight of an osmotic pressure regulator.

[0082] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; and 0.4 to 1.0 parts by weight of an osmotic pressure regulator.

[0083] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; and 0.4 to 1.0 parts by weight of sodium chloride.

[0084] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.5 to 6.0 parts by weight of recombinant type III human collagen; and 0.8 to 1.0 parts by weight of sodium chloride.

[0085] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 1.0 to 10 parts by weight of recombinant type III human collagen; 0.1 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 5.0 parts by weight of a buffer.

[0086] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; 0.4 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 2.0 parts by weight of a buffer.

[0087] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; 0.4 to 1.0 parts by weight of sodium chloride; and 0.1 to 2.0 parts by weight of disodium hydrogen phosphate or PB.

[0088] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; 0.4 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 1.0 parts by weight of a buffer.

[0089] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; 0.4 to 1.0 parts by weight of sodium chloride; and 0.1 to 1.0 parts by weight of disodium hydrogen phosphate or PB.

[0090] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 7.0 parts by weight of recombinant type III human collagen; 0.7 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 0.3 parts by weight of a buffer.

[0091] According to an embodiment of the present invention, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant type III human collagen; 0.7 to 1.0 parts by weight of sodium chloride; and 0.1 to 0.3 parts by weight of disodium hydrogen phosphate or PB.

[0092] Preparation method of recombinant type III human collagen hydrogel

[0093] In the second aspect of the present invention, the present invention proposes a method for preparing the recombinant type III human collagen hydrogel described in the first aspect. According to an embodiment of the present invention, the method comprises: subjecting the recombinant type III human collagen, an osmotic pressure regulator, water, and optionally a buffer to a first mixing treatment to obtain a recombinant type III human collagen hydrogel. The preparation method of the recombinant type III human collagen hydrogel in the present invention is simple, and no chemical cross-linking agent or enzymatic agent is required for cross-linking during the preparation process. The reagents used are non-cytotoxic, non-allergenic, and any other safety risks. The prepared recombinant type III human collagen hydrogel product is safe, free of viral hazards, and will not cause allogeneic rejection reactions. In particular, it is not only free of cross-linking agent residues, but also has good biocompatibility and degradability, and can especially improve cell adhesion and promote cell proliferation and migration.

[0094] According to an embodiment of the present invention, the method further comprises: subjecting the first mixed processing product to homogenization processing.

[0095] According to an embodiment of the present invention, the homogenization process includes any one of high-pressure homogenization, high-shear homogenization, and ultrasonic homogenization.

[0096] According to an embodiment of the present invention, the homogenization treatment is carried out at a pH value of 6 to 8, preferably 6.5 to 7.5.

[0097] Biomedical materials, and use of the aforementioned recombinant type III human collagen hydrogel in preparing biomedical materials

[0098] In the third aspect of the present invention, the present invention provides a biomedical material. According to an embodiment of the present invention, the biomedical material comprises: the recombinant type III human collagen hydrogel described in the first aspect, or the recombinant type III human collagen hydrogel prepared according to the method described in the second aspect.

[0099] According to an embodiment of the present invention, the biomedical material includes any one of hydrogel fillers, hydrogel dressings and dressing patches, hydrogel scaffolds, drug sustained-release carriers, artificial skin, orthopedic repair materials, hemostatic dressings, wound repair and healing, and skin care.

[0100] In the fourth aspect of the present invention, the present invention proposes the use of the recombinant type III human collagen hydrogel described in the first aspect, or the recombinant type III human collagen hydrogel prepared according to the method described in the second aspect, in the preparation of biomedical materials.

[0101] In an optional embodiment of the present invention, the biomedical material can be used for soft tissue filling, medical aesthetics, drug delivery, etc.

[0102] According to an embodiment of the present invention, the biomedical material includes any one of hydrogel fillers, hydrogel dressings and dressing patches, hydrogel scaffolds, drug sustained-release carriers, artificial skin, orthopedic repair materials, hemostatic dressings, wound repair and healing, and skin care.

[0103] The full-length amino acid sequence encoding the recombinant type III human collagen of the present invention is shown in Table 1, and those skilled in the art can easily obtain the corresponding nucleic acid sequence.

[0104] Table 1

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0111] Example 1: Preparation of high-purity recombinant human type III collagen

[0112] The amino acid sequence of full-length recombinant type III human collagen comes from UniProtKB-P02461, which is natural type III human collagen with a total length of 1466 amino acids. After removing the N-terminal telopeptide and C-terminal telopeptide that are prone to cause immunogenicity, the sequence length is 1061 amino acids (i.e., recombinant type III human collagen), and the theoretical degree of hydroxylation is 16.9%. The amino acid sequence of full-length recombinant type III human collagen is shown in SEQ ID NO: 1, the amino acid sequence of recombinant type III human collagen without the N-terminal telopeptide and the C-terminal telopeptide is shown in SEQ ID NO: 2 (i.e., the sequence length is 1061 amino acids), the nucleotide sequence encoding the amino acid sequence of full-length recombinant type III human collagen is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the recombinant type III human collagen without the N-terminal telopeptide and the C-terminal telopeptide is shown in SEQ ID NO: 4.

[0113] In this example, a Pichia pastoris cell expression system was used, and the synthesis of the full-length recombinant human type III collagen gene sequence (i.e., the nucleotide sequence encoding the recombinant human type III collagen is shown in SEQ ID NO: 3) was entrusted to Takara Biotechnology (Dalian) Co., Ltd., and the vector construction, strain screening, and recombinant human type III collagen preparation were all completed by our company. The specific steps are as follows:

[0114] The P4H hydroxylase gene (the amino acid sequence of P4HA2 is shown in SEQ ID NO:5, and the nucleotide sequence encoding P4HA2 is shown in SEQ ID NO:6; the amino acid sequence of P4HB is shown in SEQ ID NO:7, and the nucleotide sequence encoding P4HB is shown in SEQ ID NO:8) was introduced into the pPICZ vector, and after cloning confirmation and linearization, the Pichia pastoris GS115 cells were transformed to construct chassis cells expressing P4H hydroxylase; the full-length type III human collagen gene was introduced into the pPIC3.5K vector, and after cloning confirmation and linearization, the chassis cells carrying the P4H hydroxylase gene were transformed. The clones were selected, expression screening was performed, the yeast genome was extracted, and the copy number of the P4H hydroxylase gene and the full-length type III human collagen gene was detected, wherein the copy number detection was performed by fluorescent quantitative PCR, using the double standard curve method, and the Pichia pastoris housekeeping gene GAP (Glyceraldehyde-3-phosphate dehydrogenase) was selected as the internal reference gene, and the copy number of the P4H gene and the rhC3 gene in each strain was recorded respectively. At the same time, the selected clones were expressed by shake flask fermentation. After the expression products were enzymatically cleaved of the (N, C) terminal peptides and purified in series, recombinant type III human collagen was obtained and the degree of hydroxylation was detected. The copy numbers of different genes and the ratio of the copy number of the P4H gene to the copy number of the rhC3 gene were compared, and the degree of hydroxylation of the recombinant type III human collagen expressed by the strain was combined to obtain the target strain expressing highly hydroxylated recombinant type III human collagen.

[0115] In order to obtain recombinant human type III collagen without the N-terminal telopeptide and the C-terminal telopeptide, the full-length recombinant human type III collagen was enzymatically digested with pepsin (pepsin, Sinopharm Chemical Reagent Co., Ltd.) to obtain the recombinant human type III collagen without the N-terminal telopeptide and the C-terminal telopeptide.

[0116] The SDS-PAGE profile of the recombinant type III human collagen sample with the N-terminal telopeptide and C-terminal telopeptide removed is shown in Figure 1 The HPLC spectrum of the recombinant type III human collagen sample with the N-terminal telopeptide and C-terminal telopeptide removed is as follows: Figure 2 The results of the different gene copy numbers in each strain, the ratio of the P4H gene copy number to the rhC3 gene copy number, and the hydroxylation degree of the recombinant type III human collagen expressed by the strain are shown in Table 1. Figure 1 The SDS-PAGE pattern shows that the recombinant type III human collagen sample prepared in this example, from which the N-terminal telopeptide and the C-terminal telopeptide are removed, has a triple helical structure, and its molecular weight is consistent with the theoretical value. Figure 2 As can be seen from the HPLC graph, the purity of the recombinant type III human collagen after removal of the N-terminal telopeptide and the C-terminal telopeptide is 100%.

[0117] Table 1:

[0118]

[0119]

[0120] As can be seen from the above table, by controlling the P4H gene copy number between 3.00-4.00 and the ratio of the P4H gene to the rhC3 gene copy number between (1.80-2.20):1, a relatively high hydroxylated recombinant type III human collagen can be obtained.

[0121] Therefore, by screening the P4H gene copy number and the ratio of the P4H gene and rhC3 gene copy numbers, and combining the two, a target strain of highly hydroxylated recombinant type III human collagen can be obtained.

[0122] In the following examples, the recombinant type III human collagen from which the N-terminal telopeptide and the C-terminal telopeptide are removed is referred to as recombinant type III human collagen.

[0123] Example 2: Identification of recombinant human type III collagen

[0124] In order to confirm the consistency between recombinant type III human collagen and natural type III human collagen, the recombinant type III human collagen was sequenced and the characteristic structure of the recombinant type III human collagen was characterized.

[0125] 1. Sequence determination

[0126] The recombinant type III human collagen prepared in Example 1 was sequenced and the results of identifying the N / C termini by LC / MS were as follows: Figure 3 As shown, the sequencing results show that the sequence composition and length of the recombinant type III human collagen sample are completely consistent with the amino acid sequence of natural human collagen after removing the N-terminal telopeptide and the C-terminal telopeptide (the amino acid sequence is shown in SEQ ID NO: 2).

[0127] 2. Hydroxyproline content detection

[0128] Hydroxyproline is a specific amino acid contained in collagen, and its content is positively correlated with the stability of the triple helix structure formed by collagen. The sample to be tested (i.e., the recombinant type III human collagen prepared in Example 1) is hydrolyzed into hydroxyproline at 105°C and 6 mol / L hydrochloric acid. Hydroxyproline is oxidized by chloramine T and reacts with p-dimethylaminobenzaldehyde to form a red compound, which is measured colorimetrically at a wavelength of 560 nm. The detection method is as follows:

[0129] 2.1 Solution preparation:

[0130] Hydrochloric acid solution, c(HCl) = 6 mol / L: Mix equal volumes of high-purity hydrochloric acid and water;

[0131] pH = 6.0 buffer: weigh 57g sodium acetate trihydrate, 37.5g trisodium citrate, 5.5g citric acid monohydrate, 385ml isopropanol, add 500ml water, adjust pH to 6.0 with citric acid monohydrate, and dilute to 1000ml with water;

[0132] Chloramine T solution: weigh 3.5 g of chloramine T and dilute to 50 mL with water;

[0133] Oxidant solution: Mix chloramine T solution and pH 6.0 buffer in a ratio of 1:4;

[0134] 60% perchloric acid solution: Measure 43 ml of perchloric acid and dilute to 50 ml with water;

[0135] p-Dimethylaminobenzaldehyde solution: weigh 10g p-dimethylaminobenzaldehyde and dissolve it in 15ml 60% perchloric acid solution;

[0136] Color developer: Measure 15 ml of p-dimethylaminobenzaldehyde solution and dissolve it in 65 ml of isopropanol; Sodium hydroxide solution, c(NaOH)=6 mol / L: Weigh 24 g of sodium hydroxide and dilute it to 100 ml with water.

[0137] 2.2 Preparation of L-hydroxyproline reference solution:

[0138] L-Hydroxyproline reference stock solution: Take L-Hydroxyproline reference, weigh accurately, dissolve in water and quantitatively dilute to a solution containing 20ug per 1ml;

[0139] L-Hydroxyproline reference substance series solution: Accurately measure 2.5ml, 3.75ml, 5.0ml, 7.5ml, and 10.0ml of L-Hydroxyproline reference substance stock solution into 10ml volumetric flasks respectively, make up to volume with water, and prepare L-Hydroxyproline reference substance series solution.

[0140] 2.3 Preparation of test samples: Place the sample to be tested in a hydrolysis tube, accurately weigh it, add an appropriate amount of 6 mol / L HCl, and seal the tube with nitrogen; hydrolyze at 105℃ for 22h-24h, cool it, transfer the hydrolysis product to a volumetric flask, add water to wash the hydrolysis tube, combine the washings and transfer them to a volumetric flask, add 2 drops of phenolphthalein indicator, add 6 mol / L NaOH until the solution turns pink, make up to volume with water, shake well, and prepare the test solution.

[0141] 2.4 Detection: Accurately measure 0.5 ml of blank (water), hydroxyproline reference solution series and test solution, add 1 ml of isopropanol and 0.5 ml of oxidant solution respectively, mix and place at room temperature for 4 minutes, then add 6.5 ml of color developer respectively, mix, place each tube in a 60°C water bath and heat for 15 minutes, and cool. Use blank as control, measure absorbance at 560 nm, perform linear regression on absorbance with hydroxyproline reference solution series concentration, obtain linear regression equation, and calculate hydroxyproline content in test solution, among which, Table 2 is the standard curve result of detection, and the specific standard curve diagram is shown in Figure 4 ; Table 3 is the sample test results.

[0142] Table 2

[0143] L-Hydroxyproline reference stock solution (20 μg / ml) Final concentration of L-hydroxyproline reference substance (μg / ml) <![CDATA[OD 560 ]]> Take 2.5ml of stock solution 5.0 0.180 Take 3.75ml of stock solution 7.5 0.277 Take 5ml of stock solution 10 0.372 Take 7.5ml of stock solution 15 0.543 Take 10ml of stock solution 20 0.728

[0144] Table 3

[0145] Sample batch number Hydroxyproline as a percentage of collagen JHM09CB072 16.2 JHM09CB074 16.7 JHM09CB078 16.7 JHM09CB080 16.2 JHM09CB082 16.4

[0146] Note: Table 3 shows the test results of different batches of recombinant human type III collagen.

[0147] From the results in Table 3, it can be seen that the hydroxylation degree of recombinant type III human collagen (16.2-16.7%) is basically consistent with that of natural human collagen (theoretical value 16.9%).

[0148] 3. Circular dichroic spectroscopy CD detection

[0149] The recombinant type III human collagen prepared in Example 1 was tested by circular dichroism (CD) to verify whether it has a triple helix structure. The CD spectrum of collagen has a negative peak at a wavelength near 195 nm and a positive peak at a wavelength near 221 nm. If no positive peak near 221 nm is detected, it indicates that there is no triple helix structure. The testing agency is zhejiang Shuren University, and the testing method is as follows:

[0150] 3.1 Solution preparation:

[0151] 0.5M acetic acid solution: take 1ml of acetic acid, add 33ml of purified water and mix well;

[0152] 0.05M acetic acid solution: add 1ml of 0.5M acetic acid to a 10ml volumetric flask, dilute to the mark with purified water, and mix well;

[0153] 0.06% ammonium camphorsulfonate: weigh 60 mg ammonium camphorsulfonate and dissolve it in purified water and mix well.

[0154] 3.2 The test sample preparation information is shown in Table 4.

[0155] Table 4

[0156]

[0157]

[0158] 3.3 Determination:

[0159] CD standard solution near and far UV scanning: Set the scanning wavelength to 185-340nm to scan ultrapure water for blank test, and then collect the near and far UV absorption of circular dichroism of 0.06% camphorsulfonic acid ammonium salt standard solution in the range of 185-340nm. The CD standard solution test parameters are shown in Table 5:

[0160] Table 5

[0161] Scan time per point(s) 1 Step(nm) 0.1 Bandwidth(nm) 1 Cumulative scan times 3 Wavelength scanning range 185-340nm Optical Path 1mm Scan rate 50nm / min Baseline correction Baseline CD scale 200mdeg / 0.1dOD FL scale 2000mdeg / 0.1dOD

[0162] Far UV scanning of sample solution: First scan the blank solvent (0.05M acetic acid) of the sample, then add 200μL of sample to a 1mm cuvette, perform far UV scanning at 190-260nm and collect data according to the above parameters. The sample far UV scanning parameters are shown in Table 6:

[0163] Table 6

[0164] Scan time per point(s) 4 Step(nm) 0.1 Bandwidth(nm) 1 Cumulative scan times 4 Wavelength scanning range 190-260nm Optical Path 1mm Scan rate 50nm / min Baseline correction Baseline CD scale 200mdeg / 0.1dOD FL scale 2000mdeg / 0.1dOD

[0165] All the scanned spectra were smoothed using Spectra Analysis software in Spectra Manager, and the ratio of the CD peaks and valleys of the CD standard solution near and far UV scanning analysis graphs was calculated, with the effective ratio range being 2.08±0.06.

[0166] The far UV scanning results of CD standard solution are as follows Figure 5 As shown, the ratio of the CD peaks and troughs in the far and near UV scanning analysis graph of the CD standard solution is 2.07, which meets the requirements.

[0167] The far-ultraviolet scanning results show that the recombinant type III human collagen sample meets the typical characteristics of collagen with a triple helical structure. This example exemplifies the far-ultraviolet scanning results of the JHM09-S10 sample. For details, see Figure 6 There is a negative peak near the wavelength of 195nm and a positive peak near the wavelength of 221nm, indicating that the JHM09-S10 collagen sample meets the typical characteristics of collagen with a triple helix structure, which can explain that the JHM09-S10 collagen has a triple helix structure.

[0168] Example 3: Detection of biological activity of recombinant human type III collagen

[0169] The recombinant human type III collagen (also called JHM09 stock solution or JHM09 collagen stock solution) prepared in Example 1 was subjected to biological activity tests, including cell adhesion, cell proliferation, cell migration, and the like.

[0170] Cell adhesion test: 100 μl / well of the test sample (different concentrations of porcine collagen (white solid sponge, batch number: K2224271, Shanghai Aladdin Biochemical Technology Co., Ltd.), JHM09 stock solution) and blank control (2 mM acetic acid solution) were added to a 96-well plate. Incubate overnight at 4°C, wash twice with 150 μl / well PBS on the second day, add 50 μl / well DMEM medium, collect data as background value. Then resuspend and count the cultured NIH-3T3 cells, and adjust the cell density to 2×10 6 Cells / ml, 50μl / well of cell suspension were added to 96-well plates. The 96-well plates were transferred to the 37°C, 5% CO2 incubator of the xCELLigence RTCA MP platform for culture, the detection program was started, the adhesion index was monitored for 24 hours, and the data were collected and analyzed. The results of the cell adhesion test are shown in Table 7. Comparing the results of the cell adhesion test of JHM09 collagen stock solution and porcine collagen, JHM09 collagen stock solution can promote cell adhesion, and the intensity of the effect is equivalent to that of porcine collagen.

[0171] Table 7

[0172] sample concentration Cell adhesion index JHM09 collagen stock solution 3.5mg / ml 2.0470 Porcine collagen 3.5mg / ml 2.0213 Blank control 2.0mM 0.5162

[0173] Cell proliferation detection: NIH-3T3 cells were plated in 96-well plates one day before the test, with a cell density of 1000 cells / well, and cultured in a 37°C, 5% CO2 incubator; 24 hours later, different concentrations of test groups (different concentrations of porcine collagen, JHM09 stock solution) and a blank control group (2mM acetic acid solution) were added, and 100μl CTG detection reagent was added to each well after 48 hours of culture, and the mixture was shaken (protected from light), incubated at room temperature for 30 minutes, and the chemiluminescence value was read, and the data was analyzed. The results of the cell proliferation test are shown in Table 8. Comparing the results of the JHM09 collagen stock solution and the porcine collagen cell proliferation test, the JHM09 stock solution can promote cell proliferation, and the intensity of action is equivalent to that of porcine collagen.

[0174] Table 8

[0175] sample concentration Cell proliferation (%) JHM09 collagen stock solution 3.5mg / ml 11.91 Porcine collagen 3.5mg / ml 11.99 Blank control 2.0mM -

[0176] Cell migration detection: The day before the experiment, NIH-3T3 cells were plated in a 96-well plate with a cell density of 20,000 cells / well and 100 μl / well, and cultured in a 37°C, 5% CO2 incubator; after 24 hours, the serum-free medium was replaced and cultured for 6 hours, and then scratches were made in all wells using a wound-making device, and then washed twice with 200 μl / well PBS, and then the test group (porcine collagen, JHM09 stock solution) and the blank control group (2 mM acetic acid solution) were added. The cells were photographed at different time points and the scratch width was automatically marked for analysis. The results of the cell migration detection are shown in Figure 2. Figure 7 As shown in Table 9, comparing the results of the cell migration test of JHM09 collagen stock solution and porcine collagen, JHM09 stock solution can promote cell migration, and the intensity of the effect is equivalent to that of porcine collagen.

[0177] Table 9

[0178]

[0179]

[0180] In summary, the structural characterizations of recombinant human type III collagen show that recombinant human type III collagen is highly consistent with natural type III human collagen in terms of collagen sequence composition, length, post-translational modification (hydroxylation), triple helix structure (circular dichroism spectrum CD), and assembled fiber morphology, and is equivalent to animal-derived collagen (porcine collagen) in terms of biological activity.

[0181] Example 4: Fiber self-assembly of recombinant human type III collagen

[0182] The recombinant type III human collagen prepared in Example 1 was subjected to fiber self-assembly. The recombinant type III human collagen solution was sterilized and filtered at 0.22 μm, 100 ml of the filtered sample was added to a 500 ml beaker, 100 ml of 0.6 mol / L PBS Buffer (pH 7.0) was slowly added, the pH value was adjusted to 6.6-6.8 with 1.0 mol / L NaOH, and stirred at room temperature for 20-24 h at 300-320 rpm to obtain a recombinant type III human collagen self-assembly solution. The recombinant type III human collagen self-assembly solution was evenly divided into 50 ml centrifuge tubes, centrifuged at 4 ° C and 12000 rpm for 30 min, the supernatant was discarded, and the precipitate was obtained, that is, the recombinant type III human collagen self-assembly fiber was obtained.

[0183] Dehydrated fiber preparation: The recombinant type III human collagen self-assembled fibers obtained above were treated by two different methods:

[0184] (1) Vacuum drying of collagen self-assembled fibers. Take 1 ml of collagen self-assembled fibers, add 20 ml of 0.3 mol / L PBS buffer (pH 7.0), mix roughly, homogenize and emulsify at 200-300 bar, and dispense the emulsion into 2.5 ml vials and vacuum dry.

[0185] (2) Dehydration of collagen self-assembled fibers with ethanol. Take 1 ml of collagen self-assembled fibers and dehydrate them with ethanol solutions of gradient concentrations (including 30%, 50%, 70%, 80%, 90% and 95%), treat each concentration for 15 min, and then treat twice with 100% ethanol, each time for 20 min. The sample was centrifuged at 4°C and 12,000 rpm for 30 min, the supernatant was discarded, and the precipitate was placed in a clean bench to dry naturally.

[0186] The recombinant type III human collagen self-assembled fiber samples that were vacuum dried and ethanol dehydrated (i.e., vacuum dried and dehydrated samples and ethanol dehydrated samples) were taken, gold-sprayed, and observed under a Hitachi SU-8010 scanning electron microscope. The scanning electron microscopy results were as follows: Figure 8 , Fig. 9 shown.

[0187] Depend on Figure 8 and Fig. 9 It can be seen that the recombinant type III human collagen self-assembled fibers present a typical fiber structure. In particular, the inventors found that the assembled samples obtained by the same assembly method, after different dehydration treatments, the collagen fibers present different arrangements and compact states, laying the foundation for the subsequent development of collagen-based biomaterials suitable for different application scenarios, with different mechanical strengths or as drug delivery carriers for different tissue parts.

[0188] Example 5: Composition and preparation of recombinant type III human collagen hydrogel

[0189] 1. Preparation of solvent:

[0190] 23.4% sodium chloride stock solution: Prepared with purified water, the final concentration of sodium chloride is 23.4% (g / 100mL);

[0191] 2.0% disodium hydrogen phosphate stock solution: Prepare with purified water, the final concentration of disodium hydrogen phosphate is 2.0% (g / 100mL), and the pH is adjusted to 7.0;

[0192] 10.0% citric acid (CA) stock solution: Prepared with purified water, the final concentration of citric acid is 10.0% (g / 100mL), and the pH value is adjusted to 7.0 with sodium hydroxide.

[0193] 2. Experimental group settings:

[0194] 2.1 The preparation method of recombinant type III human collagen hydrogel (hereinafter referred to as hydrogel) is as follows:

[0195] (1) Calculate the amount of recombinant type III human collagen self-assembled fibers according to the concentration and volume of the recombinant type III human collagen stock solution before assembly and the concentration and volume of the recombinant type III human collagen in the centrifugal supernatant after assembly;

[0196] (2) According to the content of each raw material in each experimental group, the recombinant type III human collagen self-assembled fibers prepared in Example 3 and the auxiliary materials (at least one of a buffer and an osmotic pressure regulator) were mixed, and the final volume was supplemented with purified water to obtain a type III human collagen self-assembled fiber mixed solution;

[0197] (3) Homogenize the above recombinant type III human collagen self-assembled fiber mixture, the homogenizer speed is 1000 rpm, the homogenization sample temperature is room temperature, the homogenization pressure is 200-300 bar, and the homogenization time is 0.5 hour to obtain the recombinant type III human collagen hydrogel. The concentration, osmotic pressure and pH value of the recombinant type III human collagen self-assembled fibers in the hydrogel were tested. The osmotic pressure (300±30mOsmol / kg) and pH value (pH7.0±0.1) of the experimental group were almost the same.

[0198] 2.2 The content of each raw material in experimental groups 1-17 is shown in Table 10. The results of the effects on the biological activity of recombinant type III human collagen hydrogel in experimental groups 1-17 are shown in Table 10:

[0199] Table 10

[0200]

[0201] Note: PB system composition: Na2HPO4+NaH2PO4.

[0202] Comparing the results of cell activity detection of gel samples with different recombinant type III human collagen fiber contents, different concentrations and compositions of buffer systems, and different concentrations of osmotic pressure regulators, it can be seen that samples containing 0.9% sodium chloride osmotic pressure regulator system, or samples containing 0.25% Na2HPO4 and 0.75% sodium chloride osmotic pressure regulator system, have relatively good biological activity in different concentrations of collagen fiber content prescriptions; as the collagen fiber concentration decreases, the cell adhesion index decreases in a concentration-dependent manner. In all experimental groups, there was no significant difference in the cell proliferation effect with different collagen fiber contents.

[0203] Example 6: Bioactivity detection of recombinant type III human collagen hydrogel

[0204] Cell adhesion detection: 100 μl / well of the test sample (different concentrations of porcine collagen (white solid sponge, batch number: K2224271, Shanghai Aladdin Biochemical Technology Co., Ltd.), the recombinant type III human collagen hydrogel prepared in Example 3 (referred to as JHM09 fiber or JHM09 collagen fiber)) and the blank control (2 mM acetic acid solution) were added to a 96-well plate. Incubate overnight at 4°C, wash twice with 150 μl / well PBS on the next day, add 50 μl / well DMEM culture medium, and collect data as background value. Then resuspend and count the cultured NIH-3T3 cells, and adjust the cell density to 2×10 6 Cells / ml, 50μl / well of cell suspension were added to 96-well plates. The 96-well plates were transferred to the 37°C, 5% CO2 incubator of the xCELLigence RTCA MP platform for culture, the detection program was started, the adhesion index was monitored for 24 hours, and the data were collected and analyzed. The results of the cell adhesion test are shown in Table 11. Comparing the results of the cell adhesion test of JHM09 collagen fiber and porcine collagen, JHM09 collagen fiber can promote cell adhesion, and the intensity of the effect is equivalent to that of porcine collagen.

[0205] Table 11

[0206] sample concentration Cell adhesion index JHM09 collagen fiber 35mg / ml (3.5%) 2.1365 Porcine collagen 35mg / ml (3.5%) 2.1359 Blank control 2.0mM 0.5133

[0207] Cell proliferation detection: NIH-3T3 cells were plated in a 96-well plate one day before the test, with a cell density of 1000 cells / well, and cultured in a 37°C, 5% CO2 incubator; after 24 hours, different concentrations of test groups (different concentrations of porcine collagen, recombinant type III human collagen hydrogel (referred to as JHM09 fiber or JHM09 collagen fiber) prepared in Example 3) were added, and after 48 hours of culture, 100 μl of CTG detection reagent was added to each well, shaken and mixed (protected from light), incubated at room temperature for 30 minutes, and the chemiluminescence value was read, and the data was analyzed. The results of the cell proliferation detection are shown in Table 12. Comparing the results of the cell proliferation test of JHM09 collagen fiber and porcine collagen, JHM09 collagen fiber can promote cell proliferation, and the intensity of action is equivalent to that of porcine collagen.

[0208] Table 12

[0209] sample concentration Cell proliferation (%) JHM09 collagen fiber 35mg / ml (3.5%) 12.1805 Porcine collagen 35mg / ml (3.5%) 12.1823 Blank control 2.0mM -

[0210] Cell migration detection: The day before the experiment, NIH-3T3 cells were plated in a 96-well plate, 20,000 cells / well, 100 μl / well, and cultured in a 37°C, 5% CO2 incubator; after 24 hours, the serum-free medium was replaced and cultured for 6 hours, and then scratches were made in all wells using a wound-making device, and then washed twice with 200 μl / well PBS. Then, the test group (porcine collagen, recombinant type III human collagen hydrogel prepared in Example 3 (referred to as JHM09 fiber or JHM09 collagen fiber)) and the blank control group (2 mM acetic acid solution) were added, and the cells were photographed at different time points and the scratch width was automatically marked for analysis. The results of the cell migration detection are shown in the figure. Fig.10 As shown in Table 13, comparing the results of the cell migration test of JHM09 collagen fibers and porcine collagen, JHM09 collagen fibers can promote cell migration, and the intensity of the effect is equivalent to that of porcine collagen.

[0211] Table 13

[0212]

[0213]

[0214] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0215] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A recombinant type III human collagen hydrogel, characterized in that: include: Recombinant human type III collagen, an osmotic pressure regulator, water, and optionally a buffer; The osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, butylene glycol, pentylene glycol, mannitol and sucrose.

2. The recombinant type III human collagen hydrogel according to claim 1, characterized in that: The amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 2; Optionally, the purity of the recombinant type III human collagen is greater than 95%; Optionally, the degree of proline hydroxylation in the recombinant type III human collagen is greater than 15.0%, preferably 16.0% to 17.5%; Optionally, the recombinant human type III collagen has a complete triple helical structure; Optionally, in the circular dichroism spectrum, the recombinant type III human collagen has a negative absorption peak at a wavelength of 190 to 200 nm and a positive absorption peak at a wavelength of 210 to 230 nm; Optionally, the recombinant type III human collagen has a negative absorption peak at a wavelength of 195 to 200 nm; Optionally, the recombinant type III human collagen has a positive absorption peak at a wavelength of 215 to 225 nm; Optionally, the circular dichroism spectrum of the recombinant type III human collagen is as shown in FIG6 ; Optionally, the recombinant human type III collagen is prepared by the following steps: Constructing a genetically engineered bacterium that expresses proline hydroxylase and full-length recombinant human type III collagen, wherein the amino acid sequence of the full-length recombinant human type III collagen is shown in SEQ ID NO: 1; Cultivating the genetically engineered bacteria, and enzymatically digesting the culture product with pepsin to obtain the recombinant type III human collagen; The copy number of proline hydroxylase in the genetically engineered bacteria is between 3.00 and 4.00, and the ratio of the copy number of proline hydroxylase to the copy number of recombinant type III human collagen is (1.80-2.20):1; Optionally, the ratio of the copy number of proline hydroxylase to the copy number of recombinant type III human collagen is (1.90-2.10):1; Optionally, based on the total volume of the recombinant type III human collagen hydrogel, the weight volume percentage of the recombinant type III human collagen is 1% to 10%.

3. The recombinant type III human collagen hydrogel according to claim 1, characterized in that: The recombinant type III human collagen is in the form of recombinant type III human collagen self-assembled fibers; Optionally, the recombinant type III human collagen self-assembled fibers are obtained by the following steps: The recombinant type III human collagen is subjected to a self-assembly treatment in a buffer solution; Optionally, the buffer solution includes at least one of phosphate buffered saline, acetate buffered saline, citrate buffered saline, sulfate buffered saline, and Tris hydrochloride buffer; Optionally, the pH value in the self-assembly treatment is 6.6 to 6.8; Optionally, the temperature of the self-assembly treatment is 25 to 35°C; Optionally, the self-assembly treatment time is 20 to 24 hours.

4. The recombinant type III human collagen hydrogel according to any one of claims 1 to 3, characterized in that: The weight ratio of the recombinant type III human collagen to the osmotic pressure regulator is (1.0-10): (0.1-1.0), preferably (3.0-8.0): (0.4-1.0); Optionally, the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol and mannitol, preferably sodium chloride; Optionally, based on the total volume of the recombinant type III human collagen hydrogel, the weight volume percentage of the osmotic pressure regulator is 0.1% to 1.0%.

5. The recombinant type III human collagen hydrogel according to any one of claims 1 to 3, characterized in that: The weight ratio of the recombinant type III human collagen, the osmotic pressure regulator and the buffer is (1.0-10.0): (0.1-1.0): (0.5-5.0), preferably (3.0-8.0): (0.4-1.0): (0.5-2.0); Optionally, the buffer is selected from at least one of sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, potassium chloride, sodium acetate, potassium acetate, citrate, sulfate and Tris hydrochloride; Optionally, the buffer is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate and citrate, preferably disodium hydrogen phosphate; Optionally, based on the total volume of the recombinant type III human collagen hydrogel, the weight volume percentage of the buffer is 0.1% to 5.0%.

6. The recombinant type III human collagen hydrogel according to any one of claims 1 to 3, characterized in that: The recombinant type III human collagen hydrogel comprises: 1.0 to 10.0 parts by weight of recombinant human type III collagen; and 0.1 to 1.0 parts by weight of an osmotic pressure regulator; Optionally, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant human type III collagen; and 0.4 to 1.0 parts by weight of an osmotic pressure regulator; Optionally, the recombinant type III human collagen hydrogel comprises: 3.0 to 8.0 parts by weight of recombinant human type III collagen; and 0.4-1.0 parts by weight of sodium chloride; Optionally, the recombinant type III human collagen hydrogel comprises: 3.5 to 6.0 parts by weight of recombinant human type III collagen; and 0.8-1.0 parts by weight of sodium chloride; Optionally, the recombinant type III human collagen hydrogel comprises: 1.0-10 parts by weight of recombinant human type III collagen; 0.1 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 5.0 parts by weight of a buffer; Optionally, the recombinant type III human collagen hydrogel comprises: 3.0-8.0 parts by weight of recombinant human type III collagen; 0.4 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 1.0 parts by weight of a buffer; Optionally, the recombinant type III human collagen hydrogel comprises: 3.0-7.0 parts by weight of recombinant human type III collagen; 0.7 to 1.0 parts by weight of an osmotic pressure regulator; and 0.1 to 0.3 parts by weight of a buffer; Optionally, the recombinant type III human collagen hydrogel comprises: 3.0-8.0 parts by weight of recombinant human type III collagen; 0.4 to 1.0 parts by weight of sodium chloride; and 0.1-1.0 parts by weight of disodium hydrogen phosphate or PB; Optionally, the recombinant type III human collagen hydrogel comprises: 3.0-8.0 parts by weight of recombinant human type III collagen; 0.7 to 1.0 parts by weight of sodium chloride; and 0.1-0.3 parts by weight of disodium hydrogen phosphate or PB.

7. A method for preparing the recombinant type III human collagen hydrogel according to any one of claims 1 to 6, characterized in that: include: The recombinant type III human collagen, an osmotic pressure regulator, water, and optionally a buffer are subjected to a first mixing treatment to obtain a recombinant type III human collagen hydrogel.

8. The method according to claim 7, characterized in that Further including: homogenizing the first mixed product; Optionally, the homogenization treatment includes any one of high-pressure homogenization, ultrasonic homogenization, and high-speed shear dispersion; Optionally, the homogenization treatment is carried out at a pH value of 6 to 8, preferably 6.5 to 7.

5.

9. A biomedical material, characterized in that: include: The recombinant type III human collagen hydrogel according to any one of claims 1 to 6, or the recombinant type III human collagen hydrogel prepared according to the method of claim 7 or 8; Optionally, the biomedical material includes any one of a hydrogel filler, a hydrogel dressing and a dressing patch, a hydrogel scaffold, a drug sustained-release carrier, an artificial skin, an orthopedic repair material, and a hemostatic dressing.

10. Use of the recombinant type III human collagen hydrogel according to any one of claims 1 to 6, or the recombinant type III human collagen hydrogel prepared according to the method of claim 7 or 8 in the preparation of biomedical materials; Optionally, the biomedical material includes any one of a hydrogel filler, a hydrogel dressing and a dressing patch, a hydrogel scaffold, a drug sustained-release carrier, an artificial skin, an orthopedic repair material, and a hemostatic dressing.

Images