Recombinant III-type human collagen dressing and preparation method thereof

Abstract

The invention discloses a recombinant III-type human collagen dressing. The recombinant III-type human collagen dressing is prepared from recombinant III-type human collagen, a thickening agent and an osmotic pressure regulator, wherein the thickening agent is selected from the group consisting of triethanolamine and carbomer; the recombinant III type human collagen exists in the form of a recombinant III type human collagen self-assembled fiber. The recombinant III type human collagen dressing provided by the invention has relatively high stability. According to the recombinant III-type human collagen dressing, no chemical modifiers, modifiers or cross-linking agents are added, the natural material characteristics of the components are kept, and all the used reagents are free of cytotoxicity, sensitization and other safety risks. Moreover, by adopting the osmotic pressure regulator, the recombinant human collagen can be prevented from producing precipitates, and the osmotic pressure regulator has good biocompatibility, degradability and high stability, and particularly has relatively high thermal stability. Therefore, by adopting the recombinant III-type human collagen dressing disclosed by the invention, healing and repairing of a wound surface can be promoted, 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 dressing and a preparation method thereof. More specifically, the present application relates to a recombinant type III human collagen dressing 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 the function of supporting organs and protecting the body in the human 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 the post-translational modifications, triple helices and other advanced structures that are unique to natural human collagen, they cannot perform all the biological functions of the triple helices of natural human collagen.

[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 dressing, collagen gel, etc. According to the source of collagen materials, collagen can be divided into animal sources and recombinant sources. Animal-derived collagen is mainly extracted from animals, such as pigs, cattle and fish, using acid or enzyme methods. Recombinant-derived 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.

[0005] At present, the mainstream collagens in the market are mostly animal-derived collagens and recombinant humanized collagens. Animal-derived collagens have complete biological activity and in vivo stability, but they have the risks of immune response and disease or virus transmission, complex ingredients, poor quality controllability, and limited sources. Although recombinant humanized collagen has overcome the shortcomings and deficiencies of animal-derived collagen, it does not have the post-translational modifications and triple helix structure unique to collagen, and lacks all the biological functions of the triple helix structure of natural human collagen, so its role and use are limited. Recombinant human 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 natural human collagen, and it is not immunogenic.

[0006] Recombinant collagen is widely used in the field of wound repair. With the increasing number of application scenarios, higher requirements are also put forward for 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 with a triple helix 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.

[0007] Therefore, there is an urgent need to develop a recombinant type III human collagen with high purity and biological effects consistent with natural human collagen and a recombinant type III human collagen dressing with high stability. Summary of the invention

[0008] 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 dressing.

[0009] This application is completed based on the following findings of the inventors:

[0010] 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.

[0011] 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 excipient molecule, resulting in precipitation when the recombinant human collagen is compounded with excipients containing such groups, or forming an unstable system. In the preparation stability study, this system will either precipitate, stratify, or produce insoluble suspended matter, thereby affecting the use of the preparation during normal storage, or affecting the shelf life, or affecting its role in 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 can easily lead to denaturation of collagen and destroy its higher-order structure, such as triple helix breakage or unwinding, and lose its biological function. The modified excipients can easily lead to changes in their physical and chemical properties, lose their characteristics as natural materials, and affect the effectiveness and safety during use.

[0012] At present, there is no dressing prepared with 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 dressing prepared by compounding full-length type III human collagen with anionic polymer excipients, and its application in the repair of non-chronic wounds or superficial wounds and the care of surrounding skin.

[0013] An ideal collagen dressing should have the following characteristics: (1) It 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) It 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. It should especially have thermal stability, which is related to the shelf life and safety of the product; (3) It can form a protective layer on the wound and act as a physical barrier.

[0014] Based on this, the inventors of the present application have experimentally explored the compounding characteristics of recombinant human collagen and anionic polymer excipients, and unexpectedly found that (1) recombinant human type III collagen is first self-assembled into collagen fibers and then compounded; (2) a mixed solution of carbomer and triethanolamine is mixed with recombinant human type III collagen self-assembled fibers in a certain proportion; (3) when glycerol is used as an osmotic pressure regulator for collagen dressings, and (4) when a pH regulator is optionally added, a stable compounding of recombinant human type III collagen and anionic polymer excipients can be achieved, precipitation can be prevented, and a highly stable compounding system can be obtained.

[0015] Therefore, in one aspect of the present application, the present application proposes a recombinant type III human collagen dressing. According to an embodiment of the present application, the recombinant type III human collagen dressing comprises: recombinant type III human collagen, a thickener, and an osmotic pressure regulator; wherein the thickener is selected from triethanolamine and carbomer. The recombinant type III human collagen dressing of the present application has high stability; the recombinant type III human collagen exists in the form of recombinant type III human collagen self-assembled fibers.

[0016] In another aspect of the present application, the present application proposes a method for preparing the above-mentioned recombinant type III human collagen dressing. According to an embodiment of the present application, the method comprises: subjecting the recombinant type III human collagen, a thickener, an osmotic pressure regulator, and optionally a pH regulator to a first mixing treatment to obtain a recombinant type III human collagen dressing. The preparation method of the present application is simple, and the prepared recombinant type III human collagen dressing has 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 dressing or the recombinant type III human collagen dressing prepared according to the above-mentioned method. The biomedical material of the present application can promote the healing and repair of wound surface and reconstruct the healthy skin ecology.

[0018] In another aspect of the present application, the present application proposes a use of the above-mentioned recombinant human type III collagen dressing, the recombinant human type III collagen dressing prepared according to the above-mentioned method, 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 3The 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 8 The 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 scanning electron microscope (SEM) image of the JHM09 recombinant type III human collagen self-assembled fiber ethanol dehydration in Example 4 of the present application is shown;

[0030] Fig.10 Shows the dressing sample images of samples 4-6 in Example 5 of the present invention;

[0031] Fig.11 Shown are the dressing sample images of Comparative 2-6 samples in Example 5 of the present invention. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] The endpoints and any values ​​of the scope disclosed in this article are not limited to the precise scope or value, and these scopes or values ​​should be understood to include values ​​close to these scopes or values. For numerical ranges, between the endpoint values ​​of each scope, between the endpoint values ​​of each scope and a separate point value, and between separate point values, one or more new numerical ranges can be combined with each other, and these numerical ranges should be considered as specifically disclosed in this article. The term "about" is used in this article to provide textual support for the precise numerical value in front of it and the numerical value before approaching or approximating the term. In determining whether a numerical value is close to or approximating a specifically stated numerical value, the approaching or approximate unstated numerical value can be such a numerical value, and in the context in which it is provided, it provides the substantial equivalence of the specifically stated numerical value. The term "about" or "approximately" refers to an acceptable error for a specific value determined by a person skilled in the art, which depends in part on how to measure or determine the value. In certain embodiments, the term "about" or "approximately" refers to within 1, 2, 3 or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

[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, JHM09 collagen stock solution or JHM09 stock solution.

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

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

[0043] Recombinant human type III collagen dressing

[0044] In one aspect of the present application, the present application proposes a recombinant type III human collagen dressing. According to an embodiment of the present application, the recombinant type III human collagen dressing comprises: recombinant type III human collagen, a thickener, and an osmotic pressure regulator; wherein the thickener is selected from triethanolamine and carbomer; the recombinant type III human collagen exists in the form of recombinant type III human collagen self-assembled fibers. The recombinant type III human collagen dressing of the present application has high stability. The recombinant type III human collagen dressing of the present application does not add any chemical modifier, modifier and cross-linking agent, maintains the natural material properties of the components, and the reagents used are non-cytotoxic, non-allergenic and other safety risks. In addition, the use of the above-mentioned osmotic pressure regulator 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 dressing of the present application can promote the healing and repair of the wound surface, reconstruct the healthy skin ecology, and provide a new choice for biomedical materials.

[0045] The inventors found in the experiment that although both NaOH and triethanolamine can play a role in thickening carbomer, the thickener composed of triethanolamine and carbomer can preferentially block the carboxyl group of carbomer (neutralization thickening + hydrogen bond thickening) compared with the thickener composed of carbomer and NaOH solution (neutralization thickening), while avoiding the increase in the ionic strength of the system caused by the addition of NaOH, which produces a destructive effect on the stable system. In addition, precipitation can be avoided by compounding recombinant human collagen and carbomer, thereby significantly improving the stability of the recombinant human collagen and carbomer in the recombinant type III human collagen composition, especially improving the thermal stability.

[0046] 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 the heterogeneity of the collagen solution and reducing immune rejection reactions.

[0047] According to an embodiment of the present application, the purity of the recombinant type III human collagen is greater than 95%.

[0048] According to the embodiments of the present application, the proline hydroxylation degree in the recombinant type III human collagen is greater than 15.0%. 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 the heterologous solution and reducing immune rejection reactions.

[0049] 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 intrachain and interchain hydrogen bonds and maintaining the stability of the triple helix structure of collagen to exert its 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.

[0050] 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 heterogeneity of the solution and reducing immune rejection reactions.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 is no cross-linking agent residue in the assembled collagen fibers, which is a dangerous component that affects health.

[0056] 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.

[0057] 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 formed triple helix structure will have poor stability, resulting in a large difference from natural human collagen, causing adverse reactions such as allergies due to structural differences, leading to immune rejection reactions.

[0058] 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 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] According to an embodiment of the present application, based on the total volume of the recombinant type III human collagen dressing, 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 dressing can be further improved.

[0064] In this article, without special explanation, the weight volume percentage or content of A refers to the percentage ratio of the weight of A to the volume of recombinant type III human collagen dressing (w / v, g / 100 ml).

[0065] In one embodiment of the present application, based on the total volume of the recombinant human type III collagen dressing, the weight volume percentage of the recombinant human type III collagen is 0.01% to 0.50%.

[0066] In one embodiment of the present application, based on the total volume of the recombinant human type III collagen dressing, the weight volume percentage of the recombinant human type III collagen is 0.01% to 0.20%.

[0067] According to an embodiment of the present invention, the recombinant type III human collagen self-assembly fiber is obtained by the following steps: the recombinant type III human collagen is subjected to a self-assembly treatment in a buffer solution. The recombinant type III human collagen self-assembly fiber is made of recombinant type III human collagen and does not contain a chemical cross-linking agent or an enzymatic agent. As a result, the recombinant type III human collagen self-assembly fiber has no cross-linking agent residue, can form a three-dimensional network structure, and has good biocompatibility and degradability.

[0068] According to an embodiment of the present invention, the buffer solution includes at least one of phosphate buffered saline, acetate buffered saline, citrate buffered saline, sulfate buffered saline, and Tris hydrochloride buffer;

[0069] According to an embodiment of the present invention, the pH value in the self-assembly process is 6.6-6.8.

[0070] According to an embodiment of the present invention, the self-assembly process is performed at room temperature.

[0071] According to an embodiment of the present invention, the temperature of the self-assembly treatment is 25°C to 35°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C and any two points therebetween as the range between endpoint values.

[0072] According to an embodiment of the present invention, the self-assembly process lasts for 20 hours to 24 hours.

[0073] According to an embodiment of the present invention, after the self-assembly process, the self-assembly product is subjected to a dehydration process.

[0074] According to an embodiment of the present invention, the dehydration treatment is performed by vacuum drying or ethanol dehydration.

[0075] In an optional embodiment of the present application, the carbomer is carbomer 941.

[0076] According to an embodiment of the present application, the weight ratio of the carbomer to triethanolamine is (1-2): 1. Thus, the stability of the recombinant type III human collagen composition can be further improved.

[0077] According to an embodiment of the present application, based on the total volume of the recombinant type III human collagen dressing, the weight volume percentage of the thickener is 0.05% to 1.0%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0% and others. Any two point values ​​between them are used 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.

[0078] According to an embodiment of the present application, based on the total volume of the recombinant type III human collagen dressing, the weight volume percentage of the thickener is 0.2% to 0.4%.

[0079] According to an embodiment of the present application, the weight ratio of the recombinant human type III collagen, carbomer and triethanolamine is (0.01-1.0):(0.1-0.5):(0.1-0.5), preferably (0.05-0.2):(0.1-0.2):(0.1-0.2).

[0080] According to an embodiment of the present application, the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, butylene glycol, pentylene glycol, mannitol, and sucrose.

[0081] According to an embodiment of the present application, the osmotic pressure regulator is selected from at least one of glycerol and mannitol.

[0082] According to an embodiment of the present application, the osmotic pressure regulator is selected from glycerol and mannitol.

[0083] According to an embodiment of the present application, the osmotic pressure regulator is selected from glycerol.

[0084] According to an embodiment of the present application, based on the total volume of the recombinant type III human collagen dressing, the weight volume percentage of the osmotic pressure regulator is 0.5% to 5.0%, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7. .6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0% and any two point values ​​therebetween as the range between the endpoint values, such as 1.5% ~ 3.0%, 2.0% ~ 2.5%.

[0085] According to an embodiment of the present application, based on the total volume of the recombinant type III human collagen dressing, the weight volume percentage of the osmotic pressure regulator is 1.0% to 5.0%.

[0086] According to an embodiment of the present application, the weight volume percentage of the osmotic pressure regulator is 2.0% to 3.0% based on the total volume of the recombinant type III human collagen dressing, thereby further improving the stability of the recombinant type III human collagen dressing.

[0087] According to an embodiment of the present application, a pH adjuster is further included.

[0088] According to an embodiment of the present application, the pH regulator is selected from at least one of sodium hydroxide, potassium hydroxide, arginine, phosphate, and acetate.

[0089] According to an embodiment of the present application, the pH regulator is selected from at least one of sodium hydroxide, arginine, and phosphate.

[0090] According to an embodiment of the present application, the pH adjuster is selected from phosphates.

[0091] According to an embodiment of the present application, the pH value of the recombinant type III human collagen dressing is 6.0-7.0.

[0092] According to an embodiment of the present application, the recombinant type III human collagen dressing comprises:

[0093] About 0.05 to 0.3 parts by weight of recombinant type III human collagen self-assembled fibers,

[0094] About 0.15 to 0.2 parts by weight of Carbomer,

[0095] About 0.15 to 0.2 parts by weight of triethanolamine, and

[0096] About 2.0 to 2.5 parts by weight of glycerin.

[0097] Carbomer is an anionic polymer containing a large number of carboxyl groups. When combined with highly hydroxylated recombinant human type III collagen, the recombinant human type III collagen and carbomer are very likely to produce insoluble substances such as precipitation, which seriously limits its application in biological materials or as medical dressings. However, by using the recombinant human type III collagen composition of the present application, the above combination is used to preferentially block the carboxyl groups to avoid precipitation caused by the compounding of recombinant human collagen and carbomer. Thus, the stability of the recombinant human type III collagen composition containing recombinant human collagen and carbomer can be further improved, especially the thermal stability can be improved.

[0098] According to an embodiment of the present application, the recombinant type III human collagen dressing comprises:

[0099] About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers,

[0100] About 0.1 parts by weight of Carbomer,

[0101] About 0.1 parts by weight of triethanolamine, and

[0102] About 2.0 to 2.5 parts by weight of glycerin.

[0103] Carbomer is an anionic polymer containing a large number of carboxyl groups. When combined with highly hydroxylated recombinant human type III collagen, the recombinant human type III collagen and carbomer are very likely to produce insoluble substances such as precipitation, which seriously limits its application in biological materials or as medical dressings. However, by using the recombinant human type III collagen composition of the present application, the above combination is used to preferentially block the carboxyl groups to avoid precipitation caused by the compounding of recombinant human collagen and carbomer. Thus, the stability of the recombinant human type III collagen composition containing recombinant human collagen and carbomer can be further improved, especially the thermal stability can be improved.

[0104] According to an embodiment of the present application, the recombinant type III human collagen dressing comprises:

[0105] About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers,

[0106] About 0.1 to 0.2 parts by weight of Carbomer,

[0107] About 0.1 to 0.2 parts by weight of triethanolamine,

[0108] about 0.15 parts by weight of glycerol, and

[0109] About 0.15 parts by weight of mannitol.

[0110] Carbomer is an anionic polymer containing a large number of carboxyl groups. When combined with highly hydroxylated recombinant human type III collagen, the recombinant human type III collagen and carbomer are very likely to produce insoluble substances such as precipitation, which seriously limits its application in biological materials or as medical dressings. However, by using the recombinant human type III collagen composition of the present application, the above combination is used to preferentially block the carboxyl groups to avoid precipitation caused by the compounding of recombinant human collagen and carbomer. Thus, the stability of the recombinant human type III collagen composition containing recombinant human collagen and carbomer can be further improved, especially the thermal stability can be improved.

[0111] According to an embodiment of the present application, the recombinant type III human collagen dressing comprises:

[0112] About 0.05 to 0.3 parts by weight of recombinant type III human collagen self-assembled fibers,

[0113] About 0.15 to 0.2 parts by weight of Carbomer,

[0114] About 0.15 to 0.2 parts by weight of triethanolamine,

[0115] about 2.0 to 2.5 parts by weight of glycerol, and

[0116] A pH regulator is selected from phosphate, and the pH value of the recombinant type III human collagen dressing is 6.0-7.0.

[0117] According to an embodiment of the present application, the recombinant type III human collagen dressing comprises:

[0118] About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers,

[0119] About 0.1 parts by weight of Carbomer,

[0120] About 0.1 parts by weight of triethanolamine,

[0121] about 2.0 to 2.5 parts by weight of glycerol, and

[0122] A pH regulator is selected from phosphate, and the pH value of the recombinant type III human collagen dressing is 6.0-7.0.

[0123] According to an embodiment of the present application, the recombinant type III human collagen dressing comprises:

[0124] About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers,

[0125] About 0.1 to 0.2 parts by weight of Carbomer,

[0126] About 0.1 to 0.2 parts by weight of triethanolamine,

[0127] About 0.15 parts by weight of glycerol,

[0128] about 0.15 parts by weight of mannitol, and

[0129] A pH regulator is selected from phosphate, and the pH value of the recombinant type III human collagen dressing is 6.0-7.0.

[0130] Preparation method

[0131] In the second aspect of the present application, the present application proposes a method for preparing the recombinant type III human collagen dressing described in the first aspect. According to an embodiment of the present application, the method comprises: subjecting the recombinant type III human collagen, thickener, osmotic pressure regulator, and optionally pH regulator to a first mixing treatment to obtain a recombinant type III human collagen dressing. The preparation method of the present application is simple, and no chemical cross-linking agent or enzymatic cross-linking agent, modifier or modifier is added during the preparation process to obtain a stable recombinant type III human collagen dressing. The reagents used are free of any safety risks such as cytotoxicity and sensitization. The prepared recombinant type III human collagen dressing product is safe, free of viral hazards, and will not cause secondary infection or secondary infection of the wound surface, especially will not cause allergic risks caused by residues of cross-linking agents, modifiers and modifiers, and also has good biocompatibility, degradability and high stability, especially has high thermal stability.

[0132] 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 to a first homogenization to obtain a self-assembled fiber solution; subjecting the self-assembled fiber solution and a mixture of carbomer and triethanolamine to a second mixing treatment to obtain a first mixed solution; subjecting glycerol or optional mannitol to a third mixing treatment with the first mixed solution to obtain a second mixed solution; and adjusting the pH value of the second mixed solution to 6.0-7.0 to obtain a recombinant type III human collagen dressing.

[0133] According to an embodiment of the present application, before the second mixing process, carbomer and triethanolamine are mixed in equal volumes in advance and homogenized for the second time.

[0134] According to an embodiment of the present application, the method further comprises performing a third homogenization on the recombinant type III human collagen dressing stock solution with a pH value of 6.0-7.0 to obtain a recombinant type III human collagen dressing.

[0135] According to an embodiment of the present application, the homogenization pressure of the first homogenization treatment is 100-200 bar, and the homogenization time is 0.5 to 1 hour.

[0136] According to an embodiment of the present application, the homogenization pressure of the second homogenization treatment is 50-100 bar, and the homogenization time is 0.5 to 1 hour.

[0137] According to an embodiment of the present application, the homogenization pressure of the third homogenization treatment is 100-200 bar, and the homogenization time is 0.5 to 1 hour.

[0138] According to an embodiment of the present application, the method further comprises subjecting the first homogenized product, the second homogenized product and the third homogenized product to vacuum treatment, wherein the vacuum degree of the vacuum system is less than 1 KPa.

[0139] 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.

[0140] According to an embodiment of the present application, before the second mixing process, the carbomer is sterilized by moist heat and the triethanolamine is sterilized by filtration.

[0141] According to an embodiment of the present application, before the third mixing process, the glycerol solution is filtered and sterilized in advance.

[0142] According to the method of the embodiment of the present application, in the process of preparing recombinant human type III collagen dressing, the recombinant human type III collagen is filtered and sterilized, thereby completely retaining the entire biological activity of the recombinant human type III collagen. Compared with any other sterilization method, such as moist heat sterilization, radiation sterilization, etc., its primary and secondary structures are not destroyed, and the collagen chain breakage and unwinding (destruction of the triple helix structure) do not occur, so its biological effects in the body can be fully exerted.

[0143] Biomedical Materials

[0144] In the third 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 recombinant type III human collagen dressing described in the first aspect, or the recombinant type III human collagen dressing prepared according to the method described in the second aspect. The biomedical material of the present application can promote the healing and repair of wounds and reconstruct healthy skin ecology.

[0145] 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.

[0146] use

[0147] In the fourth aspect of the present application, the present application proposes the use of the recombinant type III human collagen dressing described in the first aspect, the recombinant type III human collagen dressing prepared according to the method described in the second aspect, or the biomedical material described in the third aspect in a product, wherein the product is used to treat chronic wounds and non-chronic wounds.

[0148] In some optional embodiments of the present application, the present application proposes the use of the recombinant human type III collagen dressing described in the first aspect, the recombinant human type III collagen dressing prepared according to the method described in the second aspect, or the biomedical material described in the third aspect in the treatment of chronic wounds and / or non-chronic wounds.

[0149] In some optional embodiments of the present application, the present application proposes a recombinant human type III collagen dressing as described in the first aspect, a recombinant human type III collagen dressing prepared according to the method described in the second aspect, or the biomedical material as described in the third aspect, for use in chronic wound treatment and / or non-chronic wound treatment.

[0150] 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.

[0151] 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".

[0152] Methods for treating chronic and non-chronic wounds

[0153] In the fifth 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 dressing described in the first aspect, the recombinant type III human collagen dressing prepared according to the method described in the second aspect, or the biomedical material described in the third aspect.

[0154] 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.

[0155] 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.

[0156] Example 1: Preparation of high-purity recombinant human type III collagen

[0157] 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.

[0158] The amino acid sequence of full-length recombinant human type III collagen (hereinafter referred to as rhC3):

[0159]

[0160] Amino acid sequence of recombinant human type III collagen (removing N and C terminal peptides, hereinafter referred to as rhC_3):

[0161]

[0162] The base sequence encoding the amino acid sequence of full-length recombinant type III human collagen:

[0163]

[0164] The base sequence encoding the amino acid sequence of the recombinant type III human collagen minus the (N, C) terminal peptide:

[0165]

[0166] 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:

[0167] 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, yeast genome was extracted, and the copy number of P4H hydroxylase gene and full-length type III human collagen gene was detected. The copy number detection was performed by fluorescence quantitative PCR, using the double standard curve method, and the internal reference gene was the Pichia pastoris housekeeping gene GAP (Glyceraldehyde-3-phosphate dehydrogenase). The copy number of P4H gene and rhC3 gene (encoding full-length recombinant type III human collagen) in each strain was recorded. 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 peptide and purified in series to obtain recombinant type III human collagen, and the degree of hydroxylation was detected. The copy numbers of different genes and the ratio of the copy number of the P4H gene to the copy number of the rhC3 gene were compared, combined with the degree of hydroxylation of the recombinant type III human collagen expressed by the strain. The results of the copy numbers of different genes and the ratio of the copy number of the P4H gene to the copy number of the rhC3 gene in each strain, combined with the degree of hydroxylation of the recombinant type III human collagen expressed by the strain are shown in Table 1, and finally the target strain expressing highly hydroxylated recombinant type III human collagen was obtained.

[0168] Amino acid sequence of P4HA2:

[0169] MKLWVSALLMAWFGVLSCVQAEFFTSIGHMTDLIYAEKELVQSLKEYILVEEAKLSKIKSWANKMEALTSKSAADAEGYLAHPVNAYKLVKRLNTDWPALEDLVLQDSAAGFIANLSVQRQFFPTDEDEIGAAKALMRLQDTYRLDPGTISRGELPGTKYQAMLSVDDCFGMGRSAYNEGDYYHTVLWMEQVLKQLDAGEEATTTKSQVLDYLSYAVFQLGDLHRALELTRRLLSLDPSHERAGGNLRYFEQLLEEEREKTLTNQTEAELATPEGIYERPVDYLPERDVYESLCRGEGVKLTPRRQKRLFCRYHHGNRAPQLLIAPFKEEDEWDSPHIVRYYDVMSDEEIERIKEIAKPKLARATVRDPKTGVLTVASYRVSKSSWLEEDDDPVVARVNRRMQHITGLTVKTAELLQVANYGVGGQYEPHFDFSRNDERDTFKHLGTGNRVATFLNYMSDVEAGGATVFPDLGAAIWPKKGTAVFWYNLLRSGEGDYRTRHAACPVLVGCKWVSNKWFHERGQEFLRPCGSTEVD(SEQ ID NO:5);

[0170] Nucleotide sequence of P4HA2:

[0171]

[0172] Amino acid sequence of P4HB:

[0173] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL(SEQ ID NO:7);

[0174] Nucleotide sequence of P4HB:

[0175]

[0176] 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 (i.e., rhC_3).

[0177] 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%.

[0178] Table 1:

[0179]

[0180] 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.

[0181] 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.

[0182] 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.

[0183] Example 2: Identification of recombinant human type III collagen

[0184] 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.

[0185] 1. Sequence determination

[0186] 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).

[0187] 2. Hydroxyproline content detection

[0188] 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:

[0189] 2.1 Solution preparation:

[0190] Hydrochloric acid solution, c(HCl) = 6 mol / L: Mix equal volumes of high-purity hydrochloric acid and water;

[0191] 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;

[0192] Chloramine T solution: weigh 3.5 g of chloramine T and dilute to 50 mL with water;

[0193] Oxidant solution: Mix chloramine T solution and pH 6.0 buffer in a ratio of 1:4;

[0194] 60% perchloric acid solution: Measure 43 ml of perchloric acid and dilute to 50 ml with water;

[0195] p-Dimethylaminobenzaldehyde solution: weigh 10g p-dimethylaminobenzaldehyde and dissolve it in 15ml 60% perchloric acid solution;

[0196] 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.

[0197] 2.2 Preparation of L-hydroxyproline reference solution:

[0198] L-Hydroxyproline reference stock solution: Take L-Hydroxyproline reference, weigh accurately, dissolve in water and quantitatively dilute to a solution containing 20ug per 1ml;

[0199] 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.

[0200] 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.

[0201] 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.

[0202] Table 2

[0203] 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

[0204] Table 3

[0205] 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

[0206] Note: Table 3 shows the test results of different batches of recombinant human type III collagen.

[0207] 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%).

[0208] 3. Circular dichroic spectroscopy CD detection

[0209] 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:

[0210] 3.1 Solution preparation:

[0211] 0.5M acetic acid solution: take 1ml of acetic acid, add 33ml of purified water and mix well;

[0212] 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;

[0213] 0.06% ammonium camphorsulfonate: weigh 60 mg ammonium camphorsulfonate and dissolve it in purified water and mix well.

[0214] 3.2 The test sample preparation information is shown in Table 4.

[0215] Table 4

[0216] Sample No. S10-8-1 Recombinant human type III collagen sample concentration 0.11mg / ml Buffer 0.05M acetic acid

[0217] 3.3 Determination:

[0218] 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:

[0219] Table 5

[0220] 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

[0221] 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:

[0222] Table 6

[0223] 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

[0224] 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.

[0225] 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.

[0226] 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.

[0227] Example 3: Detection of biological activity of recombinant human type III collagen

[0228] 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.

[0229] 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.

[0230] Table 7

[0231] 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

[0232] 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.

[0233] Table 8

[0234] sample concentration Cell proliferation (%) JHM09 collagen stock solution 3.5mg / ml 11.91 Porcine collagen 3.5mg / ml 11.99 Blank control 2.0mM -

[0235] 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.

[0236] Table 9

[0237]

[0238] In summary, the structural characterizations of recombinant human type III collagen show that in terms of collagen sequence composition, length, post-translational modification (hydroxylation), triple helix structure (circular dichroism spectrum CD), etc., recombinant human type III collagen is highly consistent with natural type III human collagen, and is equivalent to animal-derived collagen (porcine collagen) in terms of biological activity.

[0239] Example 4: Fiber self-assembly of recombinant human type III collagen

[0240] 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.

[0241] Dehydrated fiber preparation: The recombinant type III human collagen self-assembled fibers obtained above were treated by two different methods:

[0242] (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.

[0243] (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.

[0244] 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.

[0245] 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.

[0246] Example 5: Preparation of highly stable recombinant human type III collagen dressing and its stability testing

[0247] 1. Preparation of recombinant human type III collagen dressing

[0248] 1.1 Solution preparation:

[0249] 50.0% glycerol solution: prepared with purified water, natural pH;

[0250] 1.2% Carbomer solution: prepared with purified water, natural pH;

[0251] 1.2% triethanolamine solution: prepared with purified water, natural pH;

[0252] 10mM PB Buffer (Phosphate buffer, Sinopharm Chemical Reagent Co., Ltd.): prepared with purified water, pH 7.0;

[0253] 20mM PB Buffer (Phosphate Buffer, Sinopharm Chemical Reagent Co., Ltd.): prepared with purified water, natural pH value;

[0254] 10% Arginine (Arg) solution: prepared with purified water, natural pH;

[0255] Except for the carbomer solution which was sterilized by moist heat, all other solutions were sterilized by filtration using a 0.2 μm filter membrane (Hangzhou Kebet Filter Equipment Co., Ltd., PVDF).

[0256] 1.2 Sample preparation:

[0257] The components and ligands in the recombinant type III human collagen dressing are shown in Table 10. The screening process of different prescription compositions includes the concentration (content) of recombinant type III human collagen, a mixed base solution of carbomer and triethanolamine (synonymous with "mixed solution of carbomer and triethanolamine"), an osmotic pressure regulator, a pH regulator, etc. The weight percentage of each component in the recombinant type III human collagen dressing refers to the weight volume percentage (g / 100ml) of each component in the recombinant type III human collagen dressing based on the total weight of the recombinant type III human collagen dressing. The final pH of all screened samples is 6.0-7.0.

[0258] A. The preparation method of the recombinant type III human collagen dressing of samples 1-1 to 1-6 is as follows:

[0259] (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;

[0260] (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 10 mM PB Buffer were mixed, the mixture was stirred, and the mixture was homogenized (the homogenization sample temperature was room temperature, the homogenization pressure was 100-200 bar, and the homogenization time was 0.5 hour) to obtain a recombinant type III human collagen self-assembled fiber solution;

[0261] (3) According to the content of each raw material in each experimental group, the self-assembled fiber solution and carbomer were mixed, homogenized (homogenized sample temperature was room temperature, homogenized pressure was 100-200 bar, and homogenized time was 0.5 hour), and vacuumed (the vacuum degree of the vacuum system was less than 1 KPa) to obtain a recombinant type III human collagen dressing;

[0262] (4) The concentration and pH value of recombinant type III human collagen self-assembled fibers in the recombinant type III human collagen dressing were tested. The content of recombinant type III human collagen in the experimental group was consistent with the designed value, and the pH value was adjusted to between 6.0 and 6.3 using NaOH.

[0263] B. The preparation method of the recombinant type III human collagen dressing of samples 2-1 to 2-7 is as follows:

[0264] (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;

[0265] (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 10 mM PB Buffer were mixed, the mixture was stirred, and the mixture was homogenized (the homogenization sample temperature was room temperature, the homogenization pressure was 100-200 bar, and the homogenization time was 0.5 hour) to obtain a recombinant type III human collagen self-assembled fiber solution;

[0266] (3) According to the content of each raw material in each experimental group, prepare carbomer and triethanolamine base liquid (prepare different concentrations of carbomer and triethanolamine base liquid based on the final prescription requirements. For example, if the final prescription composition is 0.3% carbomer + 0.3% triethanolamine, first dissolve 1.2g of carbomer in 100ml of water to prepare a 1.2% carbomer solution and dissolve it overnight. After sterilization, mix it with an equal volume of 1.2% triethanolamine to obtain 0.6% carbomer + 0.6% triethanolamine. When preparing the prescription in the subsequent step (4), mix it with an equal volume of homogenized self-assembled fibers. The carbomer content in the final sample is 0.3%, and the triethanolamine content is 0.3%), and homogenize the base liquid (the homogenization sample temperature is room temperature, the homogenization pressure is 50-100bar, and the homogenization time is 0.5 hour);

[0267] (4) according to the content of each raw material in each experimental group, the self-assembled fiber solution and the carbomer and triethanolamine base solution are mixed to obtain a first mixed solution;

[0268] (5) vacuuming the first mixed liquid homogenate with a pH value of 6.0-7.0, wherein the vacuum degree of the vacuum system is less than 1 KPa, to obtain a recombinant type III human collagen dressing;

[0269] (6) The concentration and pH value of recombinant type III human collagen self-assembled fibers in the recombinant type III human collagen dressing were tested. 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 6.3.

[0270] C. The preparation method of the recombinant type III human collagen dressing of samples 3-1 to 3-36 is as follows:

[0271] (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;

[0272] (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 10 mM PB Buffer were mixed, the mixture was stirred, and the mixture was homogenized (the homogenization sample temperature was room temperature, the homogenization pressure was 100-200 bar, and the homogenization time was 0.5 hour) to obtain a recombinant type III human collagen self-assembled fiber solution;

[0273] (3) According to the content of each raw material in each experimental group, prepare carbomer and triethanolamine base solution, and homogenize the base solution (homogeneous sample temperature is room temperature, homogenization pressure is 50-100 bar, and homogenization time is 0.5 hour);

[0274] (4) according to the content of each raw material in each experimental group, the self-assembled fiber solution and the carbomer and triethanolamine base solution are mixed to obtain a first mixed solution;

[0275] (5) adding an osmotic pressure regulator (glycerol, mannitol, NaCl) to the first mixed solution according to the content of each raw material in each experimental group, and mixing the mixture to obtain a second mixed solution;

[0276] (6) vacuuming the second mixed liquid homogenate, wherein the vacuum degree of the vacuum system is less than 1 KPa, to obtain a recombinant type III human collagen dressing;

[0277] (7) The concentration and pH value of the recombinant type III human collagen self-assembled fibers in the dressing were tested. 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 6.3.

[0278] Further, the pH value of the samples was investigated, specifically: the pH value of samples 3-9 was adjusted with NaOH, and the stability of different samples (the difference was only in the pH value) was tested. The results showed that the stability effect was better when the pH value was 6.8-7.0. Therefore, the subsequent screening of pH adjusters was carried out under the condition of pH value of 6.8-7.0.

[0279] D. The preparation method of the recombinant type III human collagen dressing of samples 4-1 to 4-9 is as follows:

[0280] (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;

[0281] (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 10 mM PB Buffer were mixed, the mixture was stirred, and the mixture was homogenized (the homogenization sample temperature was room temperature, the homogenization pressure was 100-200 bar, and the homogenization time was 0.5 hour) to obtain a recombinant type III human collagen self-assembled fiber solution;

[0282] (3) According to the content of each raw material in each experimental group, prepare carbomer and triethanolamine base solution, and homogenize the base solution (homogeneous sample temperature is room temperature, homogenization pressure is 50-100 bar, and homogenization time is 0.5 hour);

[0283] (4) according to the content of each raw material in each experimental group, the self-assembled fiber solution and the carbomer and triethanolamine base solution are mixed to obtain a first mixed solution;

[0284] (5) according to the content of each raw material in each experimental group, adding glycerol to the first mixed solution and mixing them to obtain a second mixed solution;

[0285] (6) adjusting the pH value of the second mixed solution to 6.8-7.0 and homogenizing (homogenizing sample temperature room temperature, homogenizing pressure 100-200 bar, homogenizing time 0.5 hour);

[0286] (7) vacuuming the second mixed liquid homogenate with a pH value of 6.8-7.0, wherein the vacuum degree of the vacuum system is less than 1 KPa, to obtain a recombinant type III human collagen dressing;

[0287] (8) The concentration and pH value of the recombinant type III human collagen self-assembled fibers in the dressing were tested. The content of recombinant type III human collagen in the experimental group was consistent with the designed value, and the pH value was between 6.8 and 7.0.

[0288] E. The difference between the recombinant type III human collagen dressings of sample comparison 1-1 to comparison 1-6 and samples 1-1 to 1-6 is that the recombinant type III human collagen stock solution before assembly is used to replace the recombinant type III human collagen self-assembled fibers. Other conditions are the same.

[0289] F. The difference between the recombinant type III human collagen dressings of samples 2-1 to 2-7 and samples 2-1 to 2-7 is that the recombinant type III human collagen stock solution before assembly is used to replace the recombinant type III human collagen self-assembled fibers. Other conditions are the same.

[0290] Table 10

[0291]

[0292]

[0293] Among them, carbomer (Shanghai Titan Technology Co., Ltd.); triethanolamine (Shanghai Titan Technology Co., Ltd.); glycerol (Hunan Erkang Pharmaceutical Co., Ltd.); mannitol (Merck Chemical Technology (Shanghai) Co., Ltd.) The osmotic pressure of each sample in this application is within the range of 280±20mOsm / kg.

[0294] 2. Stability test of recombinant human type III collagen dressing

[0295] Experimental steps:

[0296] The recombinant type III human collagen dressing prepared in step 1 of this example was packaged into 2 ml vials at a rate of 1.5 ml / bottle, and rubber stoppers and aluminum caps were added. The sample numbers / experimental numbers were indicated, and the vials were 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 "-".

[0297] The sample preparation completion time was recorded as 0, and the stability of samples was observed for 0, 1, 3, 5, 7, 14, 21, and 30 days respectively. For the stable samples placed at 4°C, 25°C, and 37°C and placed for 30 days, the stability was further observed for 60 days and 90 days. Among them, flocculent precipitation appeared in comparison 1-1 to comparison 1-6 and comparison 2-1 to comparison 2-7 at mixing time 0; the test results of other samples are shown in Table 11. This embodiment exemplarily shows the dressing sample pictures of samples 4-6 (4°C, 90 days), and the dressing sample pictures of comparison samples 2-6 at mixing time 0.

[0298] Table 11

[0299]

[0300]

[0301]

[0302]

[0303] From the results in Tables 10 and 11, we find that:

[0304] (1) Compared with the immediate flocculent precipitation produced by the combination of recombinant type III human collagen stock solution (non-assembled) and carbomer, the stability of the system is improved by using recombinant type III human collagen self-assembled fibers. The stability is greatly improved by using a mixed base liquid of carbomer and triethanolamine and compounding recombinant type III human collagen self-assembled fibers. In particular, the stability of the recombinant type III human collagen dressing is further improved by using a mixed base liquid prepared with a carbomer and triethanolamine ratio of 1:1.

[0305] (2) Considering that there are many factors that affect recombinant type III human collagen dressings, a mixed base liquid with a ratio of 1:1 between carbomer and triethanolamine (0.1:0.1 and 0.2:0.2, respectively) was selected for screening the composition of the osmotic pressure regulator. Compared with 0.9% NaCl as an osmotic pressure regulator, 1.5% glycerol and 1.5% mannitol, or glycerol alone as an osmotic pressure regulator, the stability of the recombinant type III human collagen dressing is stronger. Compared with 1.5% glycerol and 1.5% mannitol as an osmotic pressure regulator, using 2.2% glycerol as an osmotic pressure regulator, when the ratio of carbomer to triethanolamine is 0.2:0.2, a wider concentration range of recombinant type III human collagen can be selected, which can meet the stability requirements of recombinant type III human collagen dressings.

[0306] (3) The inventors unexpectedly discovered that selecting 2.2% glycerol as an osmotic pressure regulator not only has the effect of regulating osmotic pressure, but also has the effect of synergistically enhancing the stability and improving the stability with the pH regulator.

[0307] 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.

[0308] 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 dressing, characterized in that: including recombinant type III human collagen, a thickener, and an osmotic pressure regulator; Wherein, the thickener is selected from triethanolamine and carbomer; The recombinant type III human collagen exists in the form of recombinant type III human collagen self-assembled fibers.

2. The recombinant type III human collagen dressing according to claim 1, characterized in that: The amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 2; Optionally, the purity of the recombinant type III human collagen is greater than 95%; Optionally, the degree of proline hydroxylation in the recombinant type III human collagen is greater than 15.0%, preferably 16.0% to 17.5%; Optionally, the recombinant human type III collagen has a complete triple helical structure; Optionally, in the circular dichroism spectrum, the recombinant type III human collagen has a negative absorption peak at a wavelength of 190 to 200 nm and a positive absorption peak at a wavelength of 210 to 230 nm; Optionally, the recombinant type III human collagen has a negative absorption peak at a wavelength of 195 to 200 nm; Optionally, the recombinant type III human collagen has a positive absorption peak at a wavelength of 215 to 225 nm; Optionally, the 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 self-assembly 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 volume of the recombinant human type III collagen dressing, the weight volume percentage of the recombinant human type III collagen is 0.01% to 1.0%, preferably 0.01% to 0.50%, and more preferably 0.01% to 0.20%.

4. The recombinant type III human collagen dressing according to claim 1, characterized in that: The weight ratio of carbomer to triethanolamine is (1-2):1; Optionally, the weight volume percentage of the thickener is 0.05% to 1.0%, preferably 0.2% to 0.4%, based on the total volume of the recombinant type III human collagen dressing; Optionally, the weight ratio of the recombinant human type III collagen, carbomer and triethanolamine is (0.01-1.0):(0.1-0.5):(0.1-0.5), preferably (0.05-0.2):(0.1-0.2):(0.1-0.2).

5. The recombinant type III human collagen dressing according to claim 1, characterized in that: The osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, butylene glycol, pentylene glycol, mannitol, and sucrose; Optionally, the osmotic pressure regulator is selected from at least one of glycerol and mannitol; Optionally, the weight volume percentage of the osmotic pressure regulator is 0.5% to 5.0%, preferably 2.0% to 3.0%, based on the total volume of the recombinant type III human collagen dressing; and / or, further comprising a pH adjuster; Optionally, the pH regulator is selected from at least one of sodium hydroxide, potassium hydroxide, arginine, phosphate, and acetate; Optionally, the pH regulator is selected from at least one of sodium hydroxide, arginine, and phosphate; Optionally, the pH value of the recombinant type III human collagen dressing is 6.0-7.

0.

6. The recombinant type III human collagen dressing according to any one of claims 1 to 5, characterized in that: The recombinant type III human collagen dressing comprises: About 0.05 to 0.3 parts by weight of recombinant type III human collagen self-assembled fibers, About 0.15 to 0.2 parts by weight of Carbomer, About 0.15 to 0.2 parts by weight of triethanolamine, and About 2.0 to 2.5 parts by weight of glycerin; Optionally, the recombinant type III human collagen dressing comprises: About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers, About 0.1 parts by weight of Carbomer, About 0.1 parts by weight of triethanolamine, and About 2.0 to 2.5 parts by weight of glycerin; Optionally, the recombinant type III human collagen dressing comprises: About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers, About 0.1 to 0.2 parts by weight of Carbomer, About 0.1 to 0.2 parts by weight of triethanolamine, about 0.15 parts by weight of glycerol, and About 0.15 parts by weight of mannitol.

7. The recombinant type III human collagen dressing according to any one of claims 1 to 5, characterized in that: The recombinant type III human collagen dressing comprises: About 0.05 to 0.3 parts by weight of recombinant type III human collagen self-assembled fibers, About 0.15 to 0.2 parts by weight of Carbomer, About 0.15 to 0.2 parts by weight of triethanolamine, about 2.0 to 2.5 parts by weight of glycerol, and A pH regulator, wherein the pH regulator is selected from phosphate, and the pH value of the recombinant type III human collagen dressing is 6.0 to 7.0; Optionally, the recombinant type III human collagen dressing comprises: About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers, About 0.1 parts by weight of Carbomer, About 0.1 parts by weight of triethanolamine, about 2.0 to 2.5 parts by weight of glycerol, and A pH regulator, wherein the pH regulator is selected from phosphate, and the pH value of the recombinant type III human collagen dressing is 6.0 to 7.0; Optionally, the recombinant type III human collagen dressing comprises: About 0.05 to 0.1 parts by weight of recombinant type III human collagen self-assembled fibers, About 0.1 to 0.2 parts by weight of Carbomer, About 0.1 to 0.2 parts by weight of triethanolamine, About 0.15 parts by weight of glycerol, about 0.15 parts by weight of mannitol, and A pH regulator is selected from phosphate, and the pH value of the recombinant type III human collagen dressing is 6.0-7.

0.

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

9. A biomedical material, characterized in that: The invention comprises the recombinant type III human collagen dressing as described in any one of claims 1 to 7, or the recombinant type III human collagen dressing prepared according to the method as described in claim 8.

10. Use of the recombinant human type III collagen dressing according to any one of claims 1 to 7, the recombinant human type III collagen dressing prepared according to the method according to claim 8, or the biomedical material according to claim 9 in products for treating chronic wounds and non-chronic wounds.

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