Recombinant III-type human collagen repair liquid and preparation method thereof

Abstract

The invention discloses a recombinant III-type human collagen composition. The recombinant III-type human collagen composition comprises recombinant III-type human collagen, a first buffer solution and a stabilizer, the stabilizer is selected from at least one of glycerol, butanediol, pentanediol, mannitol and trehalose. According to the recombinant III type human collagen composition, a chemical cross-linking agent or an enzymatic promoter is not added for cross-linking, and the used reagents are free of cytotoxicity, sensitization and other safety risks. Therefore, the recombinant III-type human collagen composition has no crosslinking agent residue, can form a three-dimensional network structure, can avoid the problem that the recombinant III-type human collagen and the polysaccharide are compounded to generate precipitates, also has good biocompatibility, degradability and high stability, especially has relatively high thermal stability, and can be used for preparing the recombinant III-type human collagen composition. The wound healing and repairing can be promoted, the healthy skin ecology can be reconstructed, and a new choice is provided for a biomedical material.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and specifically to a recombinant type III human collagen repair solution and a preparation method thereof. More specifically, the present application relates to a recombinant type III human collagen composition, a recombinant type III human collagen repair solution and a preparation method thereof, biomedical materials and their uses. Background Art

[0002] Collagen is the most abundant, widely distributed and most 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. It plays a role in supporting organs and protecting the body.

[0003] The unique structural characteristics, physical and chemical properties, excellent biocompatibility and biodegradability of collagen make it an ideal biomedical material. It plays an important role in the care of non-chronic wounds or superficial wounds and surrounding skin, providing a healing environment after skin allergies, laser and photon surgery, promoting wound healing, hemostatic dressings, dermal tissue filling, artificial skin, bone repair materials, and drug sustained-release materials. In particular, it has functions and effects that other repair materials or regenerative materials cannot match in terms of inducing the production of its own collagen. Many repair materials that have been launched on the market are mostly in the form of dressings such as repair fluids or repair gels, which are composed of recombinant humanized collagen. Since they do not have advanced structures such as post-translational modifications that are unique to natural human collagen, they have no or only partial biological effects.

[0004] At the same time, collagen's good biocompatibility and degradability make it a good biomaterial choice for wound treatment as a dressing or tissue engineering scaffold, including collagen repair fluid, collagen solution, collagen sponge, collagen patch, collagen gel, etc. According to the source of collagen materials, collagen can be divided into natural and recombinant collagen. Natural collagen is mainly extracted from animals, such as pigs, cattle and fish, using acid and enzyme methods. Recombinant collagen uses genetic engineering technology to connect complete or fragmented collagen gene sequences to vectors, transform microorganisms such as Escherichia coli, yeast, or animal, plant, insect and other expression systems, and obtain them after fermentation or cultivation, separation and purification. Fragmented collagen (such as recombinant humanized collagen) does not have the post-translational modifications and triple helix structure unique to collagen, so its effects and uses are limited. Intact collagen has a full-length amino acid sequence composition consistent with natural human collagen, consistent post-translational modifications (proline hydroxylation) and triple helix structure. Its biological effects are consistent with those of natural human collagen, and it is not immunogenic, so it is widely used.

[0005] Collagen is widely used in the field of wound repair. Animal-derived collagen has complete biological activity and in vivo stability, but it has the risk of immune response and disease or virus transmission, complex ingredients, poor quality controllability, and limited sources. Recombinant collagen overcomes the shortcomings and deficiencies of animal-derived collagen and is widely used in wound repair. With the increasing number of application scenarios, higher requirements are also placed on recombinant collagen. How to make collagen have biological effects consistent with natural human collagen has become an obstacle to its application. Obtaining collagen with the correct advanced structure, especially a structure consistent with natural human collagen, has become the key to solving this problem. At the same time, it is also necessary to overcome the miscibility stability and safety of recombinant human collagen when combined or compounded with different excipients.

[0006] Therefore, there is an urgent need to develop a recombinant type III human collagen repair material with high purity and biological effects consistent with natural human collagen. Summary of the invention

[0007] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a highly stable recombinant type III human collagen repair solution.

[0008] This application is completed based on the following findings of the inventors:

[0009] Recombinant human collagen synthesized by yeast has a high-level structure consistent with natural human collagen and plays an important role in wound healing, surrounding skin tissue repair, and reconstruction of skin ecology. Recombinant human collagen synthesized by yeast has the following advantages: (1) It can effectively solve the viral hidden dangers of animal-derived collagen; (2) It is non-immunogenic; (3) It can express and prepare recombinant full-length human collagen consistent with natural human collagen; (4) It can achieve post-translational modifications (proline hydroxylation, triple helix structure, etc.) of natural human collagen; (5) It can prepare high-purity products; (6) Product quality is easy to control; (7) It can achieve good batch-to-batch consistency; (8) It can be scaled up.

[0010] Recombinant human collagen contains a large amount of hydroxylated proline, which is the basis for forming a stable triple helix structure. However, high hydroxylation also leads to some new problems. For example, the hydroxyl group on hydroxylated proline is easy to form hydrogen bonds with the hydroxyl group, carboxyl group or amine group in the polysaccharide excipients, resulting in precipitation when the recombinant human collagen is compounded with polysaccharides containing such groups, or forming an unstable system. In the preparation stability study, this system will either precipitate, stratify, or produce insoluble suspended matter, thus affecting the use of the preparation during normal storage, or affecting the shelf life, or affecting its role in treating wound healing and repair. To solve this problem, some researchers have tried to modify collagen or the excipients used, but these methods are completed under certain strong acid or strong base systems. Strong acid systems are prone to denaturation of collagen, destroying its higher-order structure and losing its biological function. The modified excipients are prone to changes in their physical and chemical properties, affecting safety during use.

[0011] At present, there is no repair fluid prepared by using recombinant full-length type III human collagen with high purity, the degree of proline hydroxylation is consistent with that of natural human collagen, and it has a complete triple helix structure and biological effects consistent with those of natural human collagen, especially the preparation of repair fluid by compounding full-length type III human collagen with polysaccharides, and its application in the repair of non-chronic wounds or superficial wounds and the care of surrounding skin.

[0012] Ideal collagen repair materials should have the following characteristics: (1) They should contain collagen with full-length structure and post-translationally modified higher-order structure, which is the basis for exerting biological effects and the prerequisite for promoting or accelerating wound healing; (2) They should have good stability, and the combination system of macromolecular materials with different characteristics should not precipitate, stratify, become turbid, or produce insoluble suspended matter, etc., and should be particularly thermally stable, which is related to the shelf life and safety of the product; (3) They should form a moist protective layer on the wound surface and have a physical barrier protection effect; (4) They should prevent infection and provide sterile protection for the wound surface.

[0013] Based on this, the inventors of the present application have experimentally explored the compounding characteristics of recombinant human collagen and polysaccharide excipients, and unexpectedly found that (1) by using recombinant human collagen to self-assemble into collagen fibers before compounding; and (2) by using glycerol and mannitol as collagen composite stabilizers, and when the two are in a certain proportion, a stable compounding of collagen and polysaccharides containing hydroxyl groups, carboxyl groups, and amino groups can be achieved, thereby obtaining a highly stable compounding system.

[0014] Therefore, in one aspect of the present application, the present application proposes a recombinant type III human collagen composition. According to an embodiment of the present application, the recombinant type III human collagen composition comprises: recombinant type III human collagen, a first buffer and a stabilizer; the stabilizer is selected from at least one of glycerol, butanediol, pentanediol, mannitol and trehalose. The recombinant type III human collagen composition according to the embodiment of the present application has high stability.

[0015] In another aspect of the present application, the present application proposes a recombinant type III human collagen repair solution. According to an embodiment of the present application, the recombinant type III human collagen repair solution comprises: 0.2 to 0.4 parts by weight of recombinant type III human collagen self-assembled fibers, 1.2 to 1.8 parts by weight of glycerol, 0.2 to 1.2 parts by weight of mannitol and / or trehalose, 0.2 to 0.4 parts by weight of carboxymethyl chitosan, and a first buffer; wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 12 to 18 mM. The recombinant type III human collagen repair solution according to the embodiment of the present application does not add chemical cross-linking agents or enzymatic agents for cross-linking, and the reagents used are free of any safety risks such as cytotoxicity and sensitization. Therefore, the recombinant type III human collagen repair solution of the present application not only has no cross-linking agent residue and can form a three-dimensional network structure, which can avoid the problem of precipitation caused by the complex of recombinant human collagen and polysaccharides, but also has good biocompatibility, degradability and high stability, especially high thermal stability, which can promote the healing and repair of wounds and reconstruct a healthy skin ecology, providing a new choice for biomedical materials.

[0016] In another aspect of the present application, the present application proposes a method for preparing the above-mentioned recombinant type III human collagen composition or the above-mentioned recombinant type III human collagen repair solution. According to an embodiment of the present application, the method comprises: mixing the recombinant type III human collagen, a stabilizer and a first buffer to obtain a recombinant type III human collagen composition. The preparation method of the present application is simple, and no chemical cross-linking agent or enzymatic agent or protein stabilizer and protein dispersant are added during the preparation process to obtain a stable collagen repair solution. The reagents used are free of any safety risks such as cytotoxicity and sensitization. The prepared recombinant type III human collagen repair solution product is safe, free of viral hazards, and will not cause secondary infection or secondary infection of the wound surface. In particular, there is no cross-linking agent residue, and there is no risk of allergies caused by cross-linking agent residues. It also has good biocompatibility, degradability and high stability, especially high thermal stability.

[0017] In another aspect of the present application, the present application proposes a biomedical material. According to an embodiment of the present application, the biomedical material includes the above-mentioned recombinant type III human collagen composition or the above-mentioned recombinant type III human collagen repair solution. The biomedical material of the present application can promote the healing and repair of wounds and reconstruct healthy skin ecology.

[0018] In another aspect of the present application, the present application proposes a use of the above-mentioned recombinant type III human collagen composition, the above-mentioned recombinant type III human collagen repair solution or the above-mentioned biomedical material in a product, wherein the product is used to treat chronic wounds and non-chronic wounds.

[0019] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0021] 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 were removed in Example 1 of the present application is shown;

[0022] Figure 2 The HPLC chart of the recombinant type III human collagen sample from which the N-terminal telopeptide and the C-terminal telopeptide were removed in Example 1 of the present application is shown;

[0023] Figure 3 The figure shows the result of the N / C-terminal identification of recombinant type III human collagen using LC / MS technology in Example 2 of the present application;

[0024] Figure 4 The standard curve of L-hydroxyproline in Example 2 of the present application is shown;

[0025] 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 application;

[0026] Figure 6 The CD spectrum of the recombinant type III human collagen sample in Example 2 of the present application is shown;

[0027] Figure 7 The results of the JHM09 recombinant type III human collagen cell migration test in Example 3 of the present application are shown;

[0028] Figure 8The vacuum drying scanning electron microscope (SEM) image of the JHM09 recombinant type III human collagen self-assembled fiber in Example 4 of the present application is shown;

[0029] Fig. 9 The ethanol dehydration scanning electron microscope (SEM) image of the JHM09 recombinant type III human collagen self-assembled fibers in Example 4 of the present application is shown. DETAILED DESCRIPTION

[0030] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0031] 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 this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0032] This application details

[0033] Definitions and general terms

[0034] 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.

[0035] In order to make the present application 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 application belongs.

[0036] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Detailed description of the recombinant type III human collagen repair solution and its preparation method

[0042] The present application proposes a recombinant type III human collagen composition, a recombinant type III human collagen repair solution and a preparation method thereof, a biomedical material and uses thereof, which will be described in detail below.

[0043] Recombinant human type III collagen composition

[0044] In the first aspect of the present application, the present application proposes a recombinant type III human collagen composition. According to an embodiment of the present application, the recombinant type III human collagen composition comprises: recombinant type III human collagen, a first buffer and a stabilizer; the stabilizer is selected from at least one of glycerol, butanediol, pentanediol, mannitol and trehalose. The composition of the present application does not add any chemical cross-linking agent or enzymatic agent, and the reagents used are non-cytotoxic, non-allergenic and any other safety risks. In addition, the use of the above-mentioned stabilizer can avoid the precipitation of recombinant human collagen output, and has good biocompatibility, degradability and high stability, especially high thermal stability. Therefore, the use of the recombinant type III human collagen composition of the present application can promote the healing and repair of wounds, reconstruct the healthy skin ecology, and provide a new choice for biomedical materials.

[0045] According to an embodiment of the present application, 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 application is 100% homologous to the natural human collagen sequence, thereby further avoiding adverse reactions such as allergies caused by heterologous repair fluid and reducing immune rejection reactions.

[0046] According to an embodiment of the present application, the purity of the recombinant type III human collagen is greater than 95%.

[0047] According to the embodiments of the present application, 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 application is close to that of natural human collagen, thereby further reducing adverse reactions such as allergies caused by heterologous repair fluids and reducing immune rejection reactions.

[0048] According to a specific embodiment of the present application, 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.

[0049] According to the embodiments of the present application, the recombinant type III human collagen has a complete triple helix structure. The recombinant type III human collagen in the present application has a complete triple helix structure, which is close to natural human collagen, thereby further avoiding adverse reactions such as allergies caused by the repair fluid due to heterogeneity and reducing immune rejection reactions.

[0050] According to an embodiment of the present application, 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.

[0051] 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.

[0052] According to an embodiment of the present application, the recombinant type III human collagen has a negative absorption peak at a wavelength of 192 to 198 nm.

[0053] According to an embodiment of the present application, the recombinant type III human collagen has a positive absorption peak at a wavelength of 215 to 225 nm.

[0054] In an optional embodiment of the present application, the recombinant type III human collagen consistent with natural type III human collagen in the present application 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.

[0055] According to an embodiment of the present application, 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.

[0056] The inventors of the present application have discovered through experiments that the number of copies of the proline hydroxylase gene and the ratio of the number of copies of the proline hydroxylase gene to the number of copies 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 number of copies of the proline hydroxylase gene is too high or too low (not between 3.00-4.00), and the ratio of the number of copies of the proline hydroxylase gene to the number of copies 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 triple helix structure formed will be poor in 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.

[0057] Therefore, the inventors of the present application unexpectedly discovered that by regulating 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, the proline hydroxylation degree of the recombinant type III human collagen can be 16.0%-17.5%, which is conducive to the formation of a triple helix structure of the recombinant type III human collagen and the maintenance of the stability of the triple helix structure. The repair solution prepared using the above recombinant type III human collagen can reduce adverse reactions such as allergies caused by structural differences and reduce immune rejection reactions.

[0058] According to an embodiment of the present application, the ratio of the copy number of proline hydroxylase to the copy number of recombinant human type III collagen is (1.90-2.10):1.

[0059] In this article, unless otherwise specified, "full-length recombinant human type III collagen" refers to a protein without removing the N-terminal peptide and the C-terminal peptide, 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 peptide and the C-terminal peptide, and its amino acid sequence is shown in SEQ ID NO: 2. In genetically engineered bacteria, the copy numbers of the full-length recombinant human type III collagen gene and the recombinant human type III collagen gene are the same.

[0060] In some embodiments of the present application, the genetically engineered bacteria are obtained by the following steps: detecting the copy number 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 whose 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.

[0061] In one embodiment of the present application, 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. For example, the 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.

[0062] According to an embodiment of the present application, the recombinant human type III collagen exists in the form of recombinant human type III collagen self-assembled fibers.

[0063] According to an embodiment of the present application, 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 second buffer. 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. Thus, 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.

[0064] According to an embodiment of the present application, the second buffer comprises at least one of a phosphate buffer, an acetate buffer, a citrate buffer, a sulfate buffer, and a Tris hydrochloride buffer;

[0065] According to an embodiment of the present application, the pH value in the self-assembly process is 6.6-6.8.

[0066] According to an embodiment of the present application, the self-assembly process is performed at room temperature.

[0067] According to an embodiment of the present application, 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 the range between any two points therebetween as endpoint values.

[0068] According to an embodiment of the present application, the time of the self-assembly treatment is 20 hours to 24 hours, for example, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours and any two point values ​​therebetween as the range between the endpoint values.

[0069] According to an embodiment of the present application, based on the total weight of the recombinant type III human collagen composition, the weight volume percentage of the recombinant type III human collagen is 0.01% to 1.0%, for example, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% and any two point values ​​therebetween as the range between the endpoint values, such as 0.01% to 0.5%. , 0.01% to 0.4%, 0.01% to 0.3%, 0.1% to 0.9%, 0.1% to 0.8%, 0.1% to 0.7%, 0.1% to 0.6%, 0.1% to 0.5%, 0.1% to 0.4%, 0.1% to 0.3%, 0.1% to 0.2%, 0.2% to 0.5%, 0.2% to 0.4%, 0.2% to 0.3%, 0.3% to 0.5%, 0.3% to 0.4%, 0.4% to 0.5%. Thus, the stability of the recombinant type III human collagen composition can be further improved.

[0070] According to an embodiment of the present application, based on the total weight of the recombinant type III human collagen composition, the weight volume percentage of the recombinant type III human collagen is 0.01% to 1.0%, such as 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% or any two point values ​​therebetween as the range value between the endpoint values, such as 0.1% to 0.5%, 0.1% to 0.4%, 0.1% to 0.3%, 0.2% to 0.5%, 0.2% to 0.4%, 0.3% to 0.5%. Thus, the stability of the recombinant type III human collagen composition can be further improved.

[0071] In this document, without special explanation, the weight volume percentage or content of A refers to the ratio of the weight of A to the volume of the recombinant human type III collagen composition (w / v, g / 100 mL).

[0072] According to an embodiment of the present application, the weight ratio of the recombinant type III human collagen and the stabilizer is (0.01-1.0): (1.0-5.0), for example, (0.01-0.5): (1.0-5.0), (0.01-0.3): (1.0-5.0), (0.1-1.0): (1.0-5.0), (0.1-0.5): (1.0-5.0), (0.01-1.0): (1.0-3.0), (0.01-0.5): (1.0-3.0), (0 .01~0.4): (1.0~3.0), (0.1~1.0): (1.0~3.0), (0.1~0.5): (1.0~3.0), (0.01~1.0): (2.0~3.0), (0.01~0.5): (2.0~3.0), (0.01~0.4): (2.0~3.0), (0.1~1.0): (2.0~3.0), (0.1~0.5): (2.0~3.0), (0.2~0.4): (2.0~3.0). In this way, the stability of the recombinant type III human collagen composition can be further improved.

[0073] According to an embodiment of the present application, the weight ratio of the recombinant type III human collagen to the stabilizer is (0.1-0.5): (1.0-5.0), preferably (0.2-0.4): (2.0-3.0). Thus, the stability of the recombinant type III human collagen composition can be further improved.

[0074] According to an embodiment of the present application, the stabilizer is selected from at least one of glycerol, mannitol and trehalose.

[0075] According to an embodiment of the present application, the stabilizer includes: glycerol; and mannitol and / or trehalose.

[0076] According to an embodiment of the present application, based on the total volume of the recombinant type III human collagen repair solution, the weight volume percentage of the stabilizer is 1.0% to 5.0%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% 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 stability of the recombinant type III human collagen repair solution can be further improved.

[0077] According to an embodiment of the present application, the weight volume percentage of the stabilizer is 2.0% to 3.0% based on the total weight of the recombinant type III human collagen composition, thereby further improving the stability of the recombinant type III human collagen repair solution.

[0078] According to an embodiment of the present application, the weight ratio of the glycerol to the mannitol and / or trehalose is (1.0-2.0):(0.5-1.5), preferably (1.2-1.8):(0.8-1.2), for example 1.5:1.0.

[0079] According to an embodiment of the present application, the stabilizer is selected from glycerol and mannitol.

[0080] According to an embodiment of the present application, the weight ratio of the glycerol to the mannitol is (1.0-2.0):(0.5-1.5), preferably (1.2-1.8):(0.8-1.2), for example, 1.5:1.0.

[0081] According to an embodiment of the present application, the stabilizer is selected from glycerol and trehalose.

[0082] According to an embodiment of the present application, the weight ratio of the glycerol to the trehalose is (1.0-2.0):(0.5-1.5), preferably (1.2-1.8):(0.8-1.2), for example, 1.5:1.0.

[0083] According to an embodiment of the present application, the first buffer is selected from at least one of phosphate buffer, acetate buffer, and citrate buffer, preferably phosphate buffer.

[0084] According to an embodiment of the present application, in the recombinant type III human collagen composition, the final concentration of the first buffer is 10-20 mM, for example, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM and the range between any two points thereof as endpoint values.

[0085] In this article, "the final concentration of the first buffer" refers to the final concentration of the active ingredient in the first buffer in the recombinant type III human collagen composition. Exemplarily, the first buffer is a phosphate buffer, and the "final concentration of the first buffer" refers to the final concentration of phosphate ions in the recombinant type III human collagen composition. In this article, without special instructions, the first buffer or the recombinant type III human collagen composition in liquid form contains water; further, as long as the content of other components in the recombinant type III human collagen composition meets the requirements, the specific content of water is not limited and is within the scope of protection of this application.

[0086] According to an embodiment of the present application, in the recombinant human type III collagen composition, the final concentration of the first buffer is 12-18 mM.

[0087] According to an embodiment of the present application, the recombinant human type III collagen composition further includes polysaccharides.

[0088] According to an embodiment of the present application, based on the total weight of the recombinant type III human collagen composition, the weight volume percentage of the polysaccharide is 0.1% to 1.0%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% or a range value between any two point values ​​as endpoint values, for example, 0.1% to 0.5%, preferably 0.2% to 0.4%.

[0089] The inventors of the present application have found that the compounding of recombinant human collagen and polysaccharides is prone to precipitation. However, under the premise of containing the above-mentioned stabilizer of the present application, precipitation of recombinant human collagen and polysaccharides (especially carboxymethyl chitosan) can be avoided, and the stability of the recombinant human collagen type III composition containing recombinant human collagen and polysaccharides can be further improved, especially the thermal stability can be improved.

[0090] According to the embodiments of the present application, the polysaccharide is selected from carboxymethyl chitosan. Carboxymethyl chitosan is an important water-soluble chitosan derivative, with good biocompatibility and biodegradability, and has the effects and effects of promoting wound healing, hemostasis, analgesia and antibacterial, and has many medical applications in the field of wound healing. Carboxymethyl chitosan belongs to polysaccharides, and contains a large number of hydroxyl groups, amino groups and carboxyl groups in the molecule. When it is combined with collagen containing the same group (especially collagen with high hydroxylation), it is very easy to produce insoluble substances such as precipitation, which seriously limits its application in biological materials. However, in the recombinant type III human collagen composition of the present application, the above-mentioned stabilizer can be used to avoid recombinant human collagen and carboxymethyl chitosan from producing precipitation. Thus, the stability of recombinant human collagen and carboxymethyl chitosan in the recombinant type III human collagen composition can be further improved, especially the thermal stability can be improved.

[0091] According to an embodiment of the present application, the molecular weight of the carboxymethyl chitosan is 100k-200kDa.

[0092] According to an embodiment of the present application, the deacetylation degree of the carboxymethyl chitosan is ≥80%.

[0093] According to an embodiment of the present application, based on the total weight of the recombinant type III human collagen composition, the weight volume percentage of the carboxymethyl chitosan is 0.1% to 1.0%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% and the range between any two points as endpoint values, such as 0.1% to 0.9%, 0.1% to 0.8%, 0.1% to 0.7%, 0.1% to 0.6%, 0.1% to 0.5%, 0.1% to 0.6%. %~0.4%, 0.1%~0.9%, 0.1%~0.8%, 0.1%~0.7%, 0.1%~0.6%, 0.1%~0.5%, 0.1%~0.4%, 0.2%~0.9%, 0.2%~0.8%, 0.2%~0.7%, 0.2%~0.6%, 0.2%~0.5%, 0.2%~0.4%, 0.2%~0.9%, 0.2%~0.8%, 0.2%~0.7%, 0.2%~0.6%, 0.2%~0.5%, 0.2%~0.4%.

[0094] According to an embodiment of the present application, based on the total weight of the recombinant human type III collagen composition, the weight volume percentage of the carboxymethyl chitosan is 0.1% to 0.5%, or 0.2% to 0.4%.

[0095] According to the embodiments of the present application, the weight ratio of the recombinant human type III collagen, stabilizer and polysaccharide is (0.01-1.0):(1.0-5.0):(0.1-1.0), preferably (0.1-0.5):(1.0-5.0):(0.1-0.5), and more preferably (0.2-0.4):(2.0-3.0):(0.2-0.4).

[0096] According to an embodiment of the present application, the recombinant human type III collagen composition is in liquid form.

[0097] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0098] 0.1 to 0.5 parts by weight of recombinant human type III collagen,

[0099] 1.0 to 5.0 parts by weight of a stabilizer, and

[0100] first buffer;

[0101] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 10-20 mM.

[0102] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0103] 0.1 to 0.5 parts by weight of recombinant human type III collagen,

[0104] 1.0 to 5.0 parts by weight of a stabilizer,

[0105] 0.1 to 0.5 parts by weight of carboxymethyl chitosan, and

[0106] first buffer;

[0107] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 10-20 mM.

[0108] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0109] 0.1 to 0.5 parts by weight of recombinant type III human collagen self-assembled fibers,

[0110] 1.0 to 2.0 parts by weight of glycerol,

[0111] 0.5-1.5 parts by weight of mannitol and / or trehalose, and

[0112] first buffer;

[0113] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 10-20 mM.

[0114] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0115] 0.1 to 0.5 parts by weight of recombinant type III human collagen self-assembled fibers,

[0116] 1.0 to 2.0 parts by weight of glycerol,

[0117] 0.5 to 1.5 parts by weight of mannitol and / or trehalose,

[0118] 0.1 to 0.5 parts by weight of carboxymethyl chitosan, and

[0119] first buffer;

[0120] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 10-20 mM.

[0121] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0122] 0.2-0.4 parts by weight of recombinant type III human collagen self-assembled fibers,

[0123] 1.2 to 1.8 parts by weight of glycerol,

[0124] 0.8 to 1.2 parts by weight of mannitol and / or trehalose,

[0125] 0.2 to 0.4 parts by weight of carboxymethyl chitosan, and

[0126] first buffer;

[0127] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 12-18 mM.

[0128] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0129] 0.2-0.4 parts by weight of recombinant type III human collagen self-assembled fibers,

[0130] 1.5 parts by weight of glycerol,

[0131] 1.0 parts by weight of mannitol and / or trehalose,

[0132] 0.3 parts by weight of carboxymethyl chitosan, and

[0133] first buffer;

[0134] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 15 mM.

[0135] Recombinant human type III collagen repair solution

[0136] In the second aspect of the present application, the present application proposes a recombinant type III human collagen repair solution. According to an embodiment of the present application, the recombinant type III human collagen repair solution comprises: 0.2 to 0.4 parts by weight of recombinant type III human collagen self-assembled fibers, 1.2 to 1.8 parts by weight of glycerol, 0.2 to 1.2 parts by weight of mannitol and / or trehalose, 0.2 to 0.4 parts by weight of carboxymethyl chitosan, and a first buffer; wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 12 to 18 mM. The recombinant type III human collagen repair solution according to the embodiment of the present application does not add chemical cross-linking agents or enzymatic agents for cross-linking, and the reagents used are free of any safety risks such as cytotoxicity and sensitization. Therefore, the recombinant type III human collagen repair solution of the present application not only has no cross-linking agent residue and can form a three-dimensional network structure, which can avoid the problem of precipitation caused by the complex of recombinant human collagen and polysaccharides, but also has good biocompatibility, degradability and high stability, especially high thermal stability, which can promote the healing and repair of wounds and reconstruct a healthy skin ecology, providing a new choice for biomedical materials.

[0137] According to an embodiment of the present application, the recombinant type III human collagen composition comprises:

[0138] 0.2-0.4 parts by weight of recombinant type III human collagen self-assembled fibers,

[0139] 1.5 parts by weight of glycerol,

[0140] 1.0 parts by weight of mannitol and / or trehalose,

[0141] 0.3 parts by weight of carboxymethyl chitosan, and

[0142] first buffer;

[0143] Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 15 mM.

[0144] Preparation method

[0145] In the third aspect of the present application, the present application proposes a method for preparing the recombinant type III human collagen composition described in the first aspect or the recombinant type III human collagen repair solution described in the second aspect. According to an embodiment of the present application, the method comprises: mixing the recombinant type III human collagen, the stabilizer and the first buffer to obtain a recombinant type III human collagen composition. The preparation method of the present application is simple, and no chemical cross-linking agent or enzymatic agent or protein stabilizer and protein dispersant are added during the preparation process to obtain a stable collagen repair solution. The reagents used are free of any safety risks such as cytotoxicity and sensitization. The prepared recombinant type III human collagen repair solution product is safe, free of viral hazards, and will not cause secondary infection or secondary infection of the wound surface. In particular, there is no cross-linking agent residue, and there is no risk of allergies caused by cross-linking agent residues. It also has good biocompatibility, degradability and high stability, especially high thermal stability.

[0146] According to an embodiment of the present application, the recombinant human type III collagen exists in the form of recombinant human type III collagen self-assembled fibers.

[0147] According to an embodiment of the present application, the mixing treatment is carried out through the following steps: subjecting the recombinant type III human collagen self-assembled fibers and the first buffer to a first premixing treatment; subjecting the stabilizer and the first buffer to a second premixing treatment; subjecting the first premixing treatment product and the second premixing treatment product to a fourth premixing treatment to obtain a recombinant type III human collagen composition.

[0148] According to an embodiment of the present application, before the fourth premixing treatment, carboxymethyl chitosan and the first buffer solution are premixed for a third time, and the third premixing product is premixed together with the first premixing product and the second premixing product for a fourth premixing treatment.

[0149] According to an embodiment of the present application, the method further comprises subjecting the product of the first premixing treatment to a first homogenization treatment before the fourth premixing treatment.

[0150] According to an embodiment of the present application, the rotation speed of the first homogenization treatment is 500-2000 rpm, the homogenization pressure is 100-200 bar, and the homogenization time is 0.5-1 hour.

[0151] According to an embodiment of the present application, the method further includes subjecting the fourth premixed product to a second homogenization process.

[0152] According to an embodiment of the present application, the method further comprises subjecting the homogenized product to a vacuum treatment.

[0153] According to an embodiment of the present application, the rotation speed of the second homogenization treatment is 500-2000 rpm, the homogenization pressure is 50-100 bar, and the homogenization time is 0.5-1 hour.

[0154] According to an embodiment of the present application, before preparing the recombinant human type III collagen self-assembly fibers, the recombinant human type III collagen is subjected to a first filtration sterilization treatment.

[0155] According to an embodiment of the present application, before the second premixing process, the stabilizer is subjected to a second filtration sterilization process.

[0156] According to an embodiment of the present application, before the second premixing treatment, the carboxymethyl chitosan is subjected to a third filtration sterilization treatment.

[0157] According to the method of the embodiment of the present application, in the process of preparing recombinant human type III collagen repair solution, all raw materials and auxiliary materials (including recombinant human type III collagen, collagen stabilizer, carboxymethyl chitosan, etc.) are sterilized by filtration, which especially retains all the biological activities of recombinant human type III collagen and its repair solution components. Compared with any other sterilization method, such as moist heat sterilization, radiation sterilization, etc., its primary and higher structures are not destroyed, collagen chain breakage and unwinding (destruction of triple helix structure) do not occur, and polysaccharide chain breakage and molecular weight changes do not occur, so its biological effects in vivo can be fully exerted.

[0158] Biomedical Materials

[0159] In the fourth aspect of the present application, the present application proposes a biomedical material. According to an embodiment of the present application, the biomedical material includes the above-mentioned recombinant type III human collagen composition or the above-mentioned recombinant type III human collagen repair solution. The biomedical material of the present application can promote the healing and repair of wounds and reconstruct healthy skin ecology.

[0160] In some optional embodiments of the present application, the biomedical material may be in liquid form, gel form, suspension form, or patch form, and the specific type is not limited, all within the scope of protection of the present application. In some optional embodiments of the present application, the biomedical material can be used for smearing, cold compress or hot compress, and can also be used in combination with a dressing patch, and the specific application method is not limited, all within the scope of protection of the present application. In some optional embodiments of the present application, the biomedical material can be used for non-chronic wounds or chronic wounds, and the specific type is not limited, all within the scope of protection of the present application.

[0161] use

[0162] In the fifth aspect of the present application, the present application proposes the use of the recombinant type III human collagen composition described in the first aspect, the recombinant type III human collagen repair solution described in the second aspect, or the biomedical material described in the fourth aspect in a product, wherein the product is used to treat chronic wounds and non-chronic wounds.

[0163] In some optional embodiments of the present application, the present application proposes the use of the recombinant type III human collagen composition described in the first aspect, the recombinant type III human collagen repair solution described in the second aspect, or the biomedical material described in the fourth aspect in the treatment of chronic wounds and / or non-chronic wounds.

[0164] In some optional embodiments of the present application, the present application proposes a recombinant type III human collagen composition as described in the first aspect, the recombinant type III human collagen repair solution as described in the second aspect, or the biomedical material as described in the fourth aspect, for use in chronic wound treatment and / or non-chronic wound treatment.

[0165] In this article, the term "chronic wound" refers to wounds that take longer to heal than normal tissue healing time, and clinically refers to wounds that have not healed after more than one month of treatment and have no tendency to heal. Exemplarily, chronic wounds include, but are not limited to, wounds caused by inflammation, ischemia, bacterial infection, etc. caused by diabetic ulcers, pressure ulcers, venous ulcers, arterial ulcers, etc.

[0166] In this article, the term "non-chronic wound" refers to an immediate, newly appeared wound. For example, non-chronic wounds include, but are not limited to, trauma caused by collision with instruments, burns, skin damage after laser or photon surgery, and other situations that do not belong to "chronic wounds".

[0167] Methods for treating chronic and non-chronic wounds

[0168] In the sixth aspect of the present application, the present application proposes a method for treating chronic wounds and non-chronic wounds. According to an embodiment of the present application, the method comprises: administering to a subject a pharmaceutically acceptable dose of the recombinant type III human collagen composition of the first aspect, the recombinant type III human collagen repair solution of the second aspect, or the biomedical material of the fourth aspect.

[0169] In some optional embodiments of the present application, the application is external use. In some optional embodiments of the present application, the biomedical material can be used for coating, cold compress or hot compress, or combined with a dressing. The specific application method is not limited and is within the scope of protection of the present application.

[0170] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0171] Example 1: Preparation of high-purity recombinant human type III collagen

[0172] 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 peptide and C-terminal peptide 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 peptide and the C-terminal peptide 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 peptide and the C-terminal peptide is shown in SEQ ID NO: 4.

[0173] Amino acid sequence of full-length recombinant human type III collagen:

[0174]

[0175] Amino acid sequence of recombinant human type III collagen (removing N and C terminal peptides):

[0176] DVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPGPPGINGSPGGGEMMPGAGIPGAPGLMGARGPGPPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESG RPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRG PTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESPGPAGPAGAPGPAGSRGAPGPQGPRGD KGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPY(SEQ ID NO:2);

[0177] The base sequence encoding the amino acid sequence of full-length recombinant type III human collagen:

[0178]

[0179] The base sequence encoding the amino acid sequence of the recombinant type III human collagen minus the (N, C) terminal peptide:

[0180]

[0181] 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. The steps of vector construction, strain screening, and preparation of recombinant human type III collagen were as follows:

[0182] 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, and the expression products were enzymatically cleaved to remove the (N, C) terminal peptides and purified in series to obtain recombinant type III human collagen, and the hydroxylation degree was detected. The different gene copy numbers and the ratio of the P4H gene copy number to the rhC3 gene copy number were compared, and the hydroxylation degree of the recombinant type III human collagen expressed by the strains was combined. Among them, the results of the different gene copy numbers and the ratio of the P4H gene copy number to the rhC3 gene copy number in each strain, and the hydroxylation degree of the recombinant type III human collagen expressed by the strains are shown in Table 1, and finally the target strain expressing highly hydroxylated recombinant type III human collagen was obtained.

[0183] Amino acid sequence of P4HA2:

[0184] MKLWVSALLMAWFGVLSCVQAEFFTSIGHMTDLIYAEKELVQSLKEYILVEEAKLSKIKSWANKMEALTSKSAADAEGYLAHPVNAYKLVKRLNTDWPALEDLVLQDSAAGFIANLSVQRQFFPTDEDEIGAAKALMRLQDTYRLDPGTISRGELPGTKYQAMLSVDDCFGMGRSAYNEGDYYHTVLWMEQVLKQLDA GEEATTTKSQVLDYLSYAVFQLGDLHRALELTRRLLSLDPSHERAGGNLRYFEQLLEEEREKTLTNQTEAELATPEGIYERPVDYLPERDVYESLCRGEGVKLTPRRQKRLFCRYHHGNRAPQLLIAPFKEEDEWDSPHIVRYYDVMSDEEIERIKEIAKPKLARATVRDPKTGVLTVASYRVSKSSWLEEDDDPVVARVNRRMQHITGLTVKTAELLQVANYGVGGQYEPHFDFSRNDERDTFKHLGTGNRVATFLNYMSDVEAGGATVFPDLGAAIWPKKGTAVFWYNLLRSGEGDYRTRHAACPVLVGCKWVSNKWFHERGQEFLRPCGSTEVD(SEQ ID NO:5);

[0185] Nucleotide sequence of P4HA2:

[0186]

[0187] Amino acid sequence of P4HB:

[0188] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL(SEQ ID NO:7);

[0189] Nucleotide sequence of P4HB:

[0190]

[0191] In order to obtain recombinant human type III collagen without N-terminal peptide and C-terminal peptide, full-length recombinant human type III collagen was cleaved with pepsin (pepsin, Sinopharm Chemical Reagent Co., Ltd.) to obtain recombinant human type III collagen without N-terminal peptide and C-terminal peptide.

[0192] In this example, the recombinant type III human collagen sample S10-8 with the N-terminal peptide and the C-terminal peptide removed was subjected to SDS-PAGE and HPLC detection. The SDS-PAGE pattern is shown in FIG. Figure 1 , its HPLC spectrum is as follows Figure 2 .Depend on Figure 1 The SDS-PAGE pattern shows that the recombinant type III human collagen sample prepared in this example without the N-terminal peptide and the C-terminal peptide 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 peptide and the C-terminal peptide is 100%.

[0193] Table 1:

[0194]

[0195] 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.

[0196] 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.

[0197] In the following examples, the recombinant type III human collagen from which the N-terminal peptide and the C-terminal peptide are removed is referred to as recombinant type III human collagen.

[0198] Example 2: Identification of recombinant human type III collagen

[0199] 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.

[0200] 1. Sequence determination

[0201] The recombinant type III human collagen S10-8 sample prepared in Example 1 was sequenced, and the results of identifying the N / C termini by LC / MS were as follows: Figure 3As 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 peptide and the C-terminal peptide (the amino acid sequence is shown in SEQ ID NO: 2).

[0202] 2. Hydroxyproline content detection

[0203] 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:

[0204] 2.1 Solution preparation:

[0205] Hydrochloric acid solution, c(HCl) = 6 mol / L: Mix equal volumes of high-purity hydrochloric acid and water;

[0206] 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;

[0207] Chloramine T solution: weigh 3.5 g of chloramine T and dilute to 50 mL with water;

[0208] Oxidant solution: Mix chloramine T solution and pH 6.0 buffer in a ratio of 1:4;

[0209] 60% perchloric acid solution: Measure 43ml of perchloric acid and dilute to 50ml with water;

[0210] p-Dimethylaminobenzaldehyde solution: weigh 10g p-dimethylaminobenzaldehyde and dissolve it in 15ml 60% perchloric acid solution;

[0211] 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.

[0212] 2.2 Preparation of L-hydroxyproline reference solution:

[0213] L-Hydroxyproline reference stock solution: Take L-Hydroxyproline reference, weigh accurately, dissolve in water and quantitatively dilute to a solution containing 20ug per 1ml;

[0214] 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.

[0215] 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.

[0216] 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.

[0217] Table 2

[0218] 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

[0219] Table 3

[0220] Sample batch number Hydroxyproline as a percentage of collagen S10-8-1 16.2 S10-8-2 16.7 S10-8-3 16.7 S10-8-4 16.2 S10-8-5 16.4

[0221] Note: Table 3 shows the test results of different batches of recombinant human type III collagen.

[0222] 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%).

[0223] 3. Circular dichroic spectroscopy CD detection

[0224] 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 Shanghai Zhongke New Life Biotechnology Co., Ltd., and the testing method is as follows:

[0225] 3.1 Solution preparation:

[0226] 0.5M acetic acid solution: take 1ml of acetic acid, add 33ml of purified water and mix well;

[0227] 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;

[0228] 0.06% ammonium camphorsulfonate: weigh 60 mg ammonium camphorsulfonate and dissolve it in purified water and mix well.

[0229] 3.2 The test sample preparation information is shown in Table 4.

[0230] Table 4

[0231] Sample No. S10-8-1 Recombinant human type III collagen sample concentration 0.11mg / ml Buffer 0.05M acetic acid

[0232] 3.3 Determination:

[0233] 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:

[0234] Table 5

[0235] 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

[0236] 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:

[0237] Table 6

[0238] 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

[0239] 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.

[0240] 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 2.06, which meets the requirements.

[0241] 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 S10-8-1 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 S10-8-1 collagen sample meets the typical characteristics of collagen with a triple helical structure. Therefore, the recombinant type III human collagen protected in this application also meets the typical characteristics of a triple helical structure.

[0242] Example 3: Detection of biological activity of recombinant human type III collagen

[0243] The recombinant type III human collagen S10-8 sample (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.

[0244] 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.

[0245] Table 7

[0246] 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

[0247] 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.

[0248] Table 8

[0249] sample concentration Cell proliferation (%) JHM09 collagen stock solution 3.5mg / ml 11.91 Porcine collagen 3.5mg / ml 11.99 Blank control 2.0mM -

[0250] 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.

[0251] Table 9

[0252]

[0253] 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.

[0254] Example 4: Fiber self-assembly of recombinant human type III collagen

[0255] The recombinant type III human collagen S10-8 sample prepared in Example 1 was subjected to fiber self-assembly. The recombinant type III human collagen S10-8 sample solution was sterilized and filtered through a 0.22 μm filter membrane (Hangzhou Kebet Filter Equipment Co., Ltd., PVDF), 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.

[0256] Dehydrated fiber preparation: The recombinant type III human collagen self-assembled fibers obtained above were treated by two different methods:

[0257] (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.

[0258] (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.

[0259] 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.

[0260] Depend on Figure 8 and Fig. 9It 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.

[0261] Example 5: Preparation of highly stable recombinant human type III collagen repair solution and its stability test

[0262] 1. Preparation of recombinant human type III collagen repair solution

[0263] 1.1 Solvent preparation:

[0264] 10.0% glycerol stock solution: prepared with purified water, natural pH value;

[0265] 12.0% mannitol stock solution: prepared with purified water, natural pH value;

[0266] 12.0% trehalose mother solution: prepared with purified water, natural pH value;

[0267] 0.6% carboxymethyl chitosan (Sinopharm Chemical Reagent Co., Ltd., molecular weight 100-200 kDa, degree of substitution ≥ 80%) mother solution: prepared with purified water, pH 7.0;

[0268] 15 mM (0.213%) PB Buffer (phosphate buffer, Sinopharm Chemical Reagent Co., Ltd.): prepared with purified water, pH 7.0;

[0269] All mother liquors were sterilized and filtered using a 0.2 μm filter membrane (Hangzhou Kebet Filter Equipment Co., Ltd., PVDF).

[0270] 1.2 Sample preparation:

[0271] The components and ligands in the recombinant type III human collagen repair solution (referred to as the repair solution) are shown in Table 10. The screening process of different prescription compositions includes differences in the concentration (content) of recombinant type III human collagen, the composition and content of the recombinant type III human collagen stabilizer (glycerol (G) / mannitol (M) / glycerol+mannitol (G+M)), and the concentration (content) of carboxymethyl chitosan (CMCS). The concentration of PB Buffer is 15mM (0.213%). The weight percentage of each component in the recombinant type III human collagen repair solution refers to the weight volume percentage (g / 100ml) of each component in the recombinant type III human collagen repair solution based on the total weight of the recombinant type III human collagen repair solution. After preparation, all screened samples were adjusted to pH 6.5-7.0 with 1.0M NaOH solution. The preparation method of the recombinant type III human collagen repair solution is as follows:

[0272] (1) Calculating 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;

[0273] (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 4 and the first buffer (PB Buffer) were first mixed to obtain a recombinant type III human collagen self-assembled fiber mixed solution, and homogenized (homogenizer speed 1000 rpm, homogenization sample temperature room temperature, homogenization pressure 100-200 bar, homogenization time 0.5 hour) to obtain a recombinant type III human collagen fiber solution A;

[0274] (3) mixing the collagen stabilizer and the first buffer for a second time to obtain solution B;

[0275] (4) mixing the carboxymethyl chitosan and the first buffer for a third time to obtain solution C;

[0276] (5) Mix the above solutions A, B, and C in order according to the prescription composition, stir and roughly mix at a stirring speed of 200-300 rpm until the system is visually uniform to obtain a recombinant type III human collagen repair solution precursor, and homogenize the repair solution precursor (homogenizer speed 1000 rpm, homogenization sample temperature room temperature, homogenization pressure 50-100 bar, homogenization time 0.5 hour) and evacuate to obtain a recombinant type III human collagen repair solution;

[0277] (6) The concentration and pH value of the recombinant type III human collagen self-assembled fibers in the repair solution were checked. The content of recombinant type III human collagen in the experimental group was consistent with the designed value, and the pH value was between 6.0 and 7.0.

[0278] Table 10

[0279]

[0280]

[0281] Among them, glycerol (G, Hunan Erkang Pharmaceutical Co., Ltd.); mannitol (M, Merck Chemical Technology (Shanghai) Co., Ltd.); butanediol (B, Nanjing Well Pharmaceutical Group Co., Ltd.); pentanediol (P, Shanghai Aladdin Biochemical Technology Co., Ltd.); trehalose (F, Hunan Erkang Pharmaceutical Co., Ltd.).

[0282] 2. Stability test of recombinant type III human collagen repair solution

[0283] 2.1 Experimental steps:

[0284] The recombinant type III human collagen repair solution prepared in step 1 of this example was dispensed into 2 ml vials at a rate of 1.5 ml / bottle, and rubber stoppers and aluminum caps were added. The samples were labeled with the sample numbers / experimental numbers, and placed in stability observation boxes at 4°C, 25°C, and 37°C, respectively. Five samples were placed in parallel in each group, and the stability of the samples at different temperatures at each time point was observed under a light detector. If there was no change in appearance compared to time 0 during the sample placement period (the sample was uniform, with no phase change (precipitation, stratification, turbidity change, etc.)), it was recorded as "+", otherwise it was recorded as "-".

[0285] The sample preparation completion time was recorded as 0, and the stability of the samples was observed at 0, 1, 3, 5, 7, 14, 21, and 30 days respectively.

[0286] 2.2 The test results are shown in Table 11.

[0287] Table 11

[0288]

[0289]

[0290]

[0291]

[0292] From the results in Tables 10 and 11, we find that:

[0293] (1) By comparing the combinations of different stabilizers in the recombinant type III collagen repair solution, it was found that the combination of glycerol and mannitol (repair solution groups 1-4 and 1-7) and the combination of glycerol and trehalose (repair solution group 1-12) had a good stabilizing effect on collagen placed at different temperatures. Considering that the stability of the complex system in the recombinant type III collagen repair solution may be affected by multiple factors, especially the composition and content of the complex system. Therefore, two different combinations of 1.5% glycerol + 1.0% mannitol (repair solution group 1-7) and 1.5% glycerol + 1.0% trehalose (repair solution group 1-12) were selected to conduct experiments on different concentrations of complex collagen and further experiments on the content (concentration) of complex carboxymethyl chitosan.

[0294] (2) The results of screening different concentrations of recombinant type III collagen in the recombinant type III collagen repair solution showed that when the concentration of recombinant type III collagen was 0.01-0.5%, it showed good stability in the 1.5% glycerol + 1.0% mannitol system; in contrast, the same recombinant type III collagen concentration showed relatively poor stability in the 1.5% glycerol + 1.0% trehalose system.

[0295] (3) The experimental results of compounding carboxymethyl chitosan with different stabilizers showed that the same concentration of carboxymethyl chitosan in the recombinant type III collagen repair solution had a better stability effect when combined with 1.5% glycerol + 1.0% mannitol than with 1.5% glycerol + 1.0% trehalose. This example unexpectedly found that the stabilizer and carboxymethyl chitosan may interfere with each other, and the two components have a synergistic effect of increasing or decreasing the effect. Therefore, the stabilizer combination protected by this application is not only applicable to the recombinant type III collagen repair solution system without carboxymethyl chitosan, but also to the recombinant type III collagen repair solution system containing carboxymethyl chitosan.

[0296] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", 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 application. 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, without contradiction.

[0297] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A recombinant type III human collagen composition, characterized in that: include: recombinant human type III collagen, a first buffer, and a stabilizer; The stabilizer is selected from at least one of glycerol, butylene glycol, pentylene glycol, mannitol and trehalose.

2. The recombinant type III human collagen composition 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 human type III 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 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 human type III collagen is (1.90-2.10):

1.

3. The recombinant type III human collagen composition according to claim 2, 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: Subjecting the recombinant type III human collagen to a self-assembly treatment in a second buffer; Optionally, the second buffer comprises at least one of a phosphate buffer, an acetate buffer, a citrate buffer, a sulfate buffer, and a 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; Optionally, based on the total weight of the recombinant human type III collagen composition, the weight volume percentage of the recombinant human type III collagen is 0.01% to 1.0%, preferably 0.1% to 0.5%.

4. The recombinant type III human collagen composition according to claim 1, characterized in that: The weight ratio of the recombinant type III human collagen to the stabilizer is (0.01-1.0):(1.0-5.0), preferably (0.1-0.5):(1.0-5.0), and more preferably (0.2-0.4):(2.0-3.0); Optionally, the stabilizer is selected from at least one of glycerol, mannitol and trehalose; Optionally, the stabilizer comprises: glycerol; and mannitol and / or trehalose; Optionally, the weight ratio of the glycerol to the mannitol and / or trehalose is (1.0-2.0):(0.5-1.5), preferably (1.2-1.8):(0.8-1.2); Optionally, the stabilizer is selected from glycerol and mannitol; Optionally, the weight ratio of the glycerol to the mannitol is (1.0-2.0):(0.5-1.5), preferably (1.2-1.8):(0.8-1.2); Optionally, the stabilizer is selected from glycerol and trehalose; Optionally, the weight ratio of the glycerol to the trehalose is (1.0-2.0):(0.5-1.5), preferably (1.2-1.8):(0.8-1.2); Optionally, the weight volume percentage of the stabilizer is 1.0% to 5.0%, preferably 2.0% to 3.0%, based on the total weight of the recombinant type III human collagen composition; Optionally, the first buffer is selected from at least one of a phosphate buffer, an acetate buffer and a citrate buffer, preferably a phosphate buffer; Optionally, in the recombinant human type III collagen composition, the final concentration of the first buffer is 10 to 20 mM, preferably 12 to 18 mM.

5. The recombinant type III human collagen composition according to claim 1, characterized in that: Further comprising polysaccharides; Optionally, the polysaccharide is selected from carboxymethyl chitosan; Optionally, based on the total weight of the recombinant type III human collagen composition, the weight volume percentage of the polysaccharide is 0.1% to 1.0%, preferably 0.1% to 0.5%, and more preferably 0.2% to 0.4%; Optionally, the weight ratio of the recombinant human type III collagen, stabilizer and polysaccharide is (0.01-1.0):(1.0-5.0):(0.1-1.0), preferably (0.1-0.5):(1.0-5.0):(0.1-0.5).

6. The recombinant human type III collagen composition according to any one of claims 1 to 5, characterized in that: The recombinant type III human collagen composition is in liquid form; Optionally, the recombinant type III human collagen composition comprises: 0.1 to 0.5 parts by weight of recombinant human type III collagen, 1.0 to 5.0 parts by weight of a stabilizer, and first buffer; Wherein, in the recombinant type III human collagen composition, the final concentration of the first buffer is 10 to 20 mM; Optionally, the recombinant type III human collagen composition comprises: 0.1 to 0.5 parts by weight of recombinant human type III collagen, 1.0 to 5.0 parts by weight of a stabilizer, 0.1 to 0.5 parts by weight of carboxymethyl chitosan, and first buffer; in, In the recombinant type III human collagen composition, the final concentration of the first buffer is 10 to 20 mM; Optionally, the recombinant type III human collagen composition comprises: 0.1 to 0.5 parts by weight of recombinant type III human collagen self-assembled fibers, 1.0 to 2.0 parts by weight of glycerol, 0.5 to 1.5 parts by weight of mannitol and / or trehalose, and first buffer; in, In the recombinant type III human collagen composition, the final concentration of the first buffer is 10 to 20 mM; Optionally, the recombinant type III human collagen composition comprises: 0.1 to 0.5 parts by weight of recombinant type III human collagen self-assembled fibers, 1.0 to 2.0 parts by weight of glycerol, 0.5 to 1.5 parts by weight of mannitol and / or trehalose, 0.1 to 0.5 parts by weight of carboxymethyl chitosan, and first buffer; in, In the recombinant type III human collagen composition, the final concentration of the first buffer is 10-20 mM.

7. A recombinant type III human collagen repair solution, characterized in that: The recombinant type III human collagen composition comprises: 0.2-0.4 parts by weight of recombinant type III human collagen self-assembled fibers, 1.2 to 1.8 parts by weight of glycerol, 0.2 to 1.2 parts by weight of mannitol and / or trehalose, 0.2 to 0.4 parts by weight of carboxymethyl chitosan, and first buffer; in, In the recombinant type III human collagen composition, the final concentration of the first buffer is 12-18 mM.

8. The recombinant type III human collagen repair solution according to claim 7, characterized in that: The recombinant type III human collagen composition comprises: 0.2-0.4 parts by weight of recombinant type III human collagen self-assembled fibers, 1.5 parts by weight of glycerol, 1.0 parts by weight of mannitol and / or trehalose, 0.3 parts by weight of carboxymethyl chitosan, and first buffer; in, In the recombinant type III human collagen composition, the final concentration of the first buffer is 15 mM.

9. A method for preparing the recombinant type III human collagen composition according to any one of claims 1 to 6 or the recombinant type III human collagen repair solution according to any one of claims 7 to 8, characterized in that: include: The recombinant type III human collagen, the stabilizer and the first buffer are mixed to obtain a recombinant type III human collagen composition.

10. The method according to claim 9, characterized in that The recombinant type III human collagen is in the form of recombinant type III human collagen self-assembled fibers; Optionally, the mixing process is carried out by the following steps: Performing a first premixing treatment on the recombinant type III human collagen self-assembled fibers and a first buffer; subjecting the stabilizer and the first buffer to a second premixing treatment; The first premixing treatment product and the second premixing treatment product are subjected to a fourth premixing treatment to obtain a recombinant type III human collagen composition; Optionally, before the fourth premixing treatment, the carboxymethyl chitosan and the first buffer solution are premixed for a third time, and the product of the third premixing treatment is premixed for a fourth time together with the product of the first premixing treatment and the product of the second premixing treatment; Optionally, the method further comprises, before the fourth premixing process, pre-homogenizing the product of the first premixing process; Optionally, the first homogenization treatment has a rotation speed of 500-2000 rpm, a homogenization pressure of 100-200 bar, and a homogenization time of 0.5-1 hour; Optionally, the method further comprises subjecting the fourth premixed product to a second homogenization process; Optionally, the method further comprises subjecting the homogenized product to a vacuum treatment; Optionally, the second homogenization treatment has a rotation speed of 500-2000 rpm, a homogenization pressure of 50-100 bar, and a homogenization time of 0.5-1 hour; Optionally, before preparing the recombinant type III human collagen self-assembly fibers, the recombinant type III human collagen is subjected to a first filtration sterilization treatment; Optionally, before the second premixing process, the stabilizer is subjected to a second filtration sterilization process in advance; Optionally, before the second premixing treatment, the carboxymethyl chitosan is subjected to a third filtration sterilization treatment.

11. A biomedical material, characterized in that: It comprises the recombinant type III human collagen composition according to any one of claims 1 to 6 or the recombinant type III human collagen repair solution according to any one of claims 7 to 8.

12. Use of the recombinant type III human collagen composition according to any one of claims 1 to 6, the recombinant type III human collagen repair solution according to any one of claims 7 to 8, or the biomedical material according to claim 11 in a product, wherein the product is used to treat chronic wounds and non-chronic wounds.

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