Recombinant collagen type iii and methods of making and using same

Abstract

The application discloses recombinant collagen type III and a preparation method and application thereof. The recombinant collagen type III comprises n structural units; the structural unit comprises peptide segments A, B and C connected in any order. The three specific sequences (A, B and C) of the selected human collagen type III are selected and connected in sequence to form the high-biological-activity recombinant collagen type III with a triple helix structure, and the recombinant collagen type III is stable in expression and easy to produce in large scale.

Description

Technical Field

[0001] This application relates to the fields of bioengineering and medical materials technology, specifically to recombinant type III collagen and its preparation method and application, and further to recombinant type III collagen, nucleic acid molecule encoding it, recombinant expression vector including the nucleic acid molecule, recombinant host cell expressing the recombinant type III collagen, method for preparing the recombinant type III collagen, and application of the recombinant type III collagen. Background Technology

[0002] Collagen is the most abundant protein in mammals, widely distributed in tissues such as skin, bones, tendons, cartilage, and blood vessels. Type III collagen, an important fibrous collagen, is mainly found in the dermis and possesses excellent repair properties, playing a crucial role in maintaining skin elasticity and promoting scar healing. With age, type III collagen is continuously lost, leading to signs of skin aging. Because its content is highest in infancy, type III collagen is also known as "infant collagen" and has received widespread attention in the market in recent years.

[0003] Currently, traditional collagen is mainly obtained by extracting it from the connective tissues of animals such as pigs, cattle, and sheep. However, this method has many shortcomings: (1) bioactivity is easily lost during the extraction process; (2) animal-derived extracts may trigger immune reactions and allergies; (3) there are significant batch-to-batch differences, and the quality is unstable. In addition, animal-derived raw materials may carry pathogens (such as prions), which seriously limits their application in the fields of biomedicine and medical aesthetics.

[0004] In recent years, advancements in synthetic biology have enabled the high-purity, high-specificity production of type III collagen without the use of animal-derived materials. However, existing recombinant type III collagen technologies still have certain limitations. For example, they lack uniqueness in sequence design, with researchers often constructing recombinant proteins based on natural full-length sequences or single fragments, resulting in insufficient biological activity and triple-helix assembly efficiency. CN119390820A discloses a recombinant humanized type III collagen, which, although employing AI technology and molecular dynamics simulations for sequence design, still has room for improvement in enhancing the stability and anti-degradation properties of the recombinant protein. Furthermore, recombinant collagen is prone to degradation during expression and production, leading to low purity (typically ≤60%) and high production costs. Currently, most functional validations are limited to cell experiments, lacking systematic evaluation using animal models, making it difficult to comprehensively demonstrate the actual efficacy of recombinant proteins in areas such as skin repair and tissue regeneration.

[0005] Therefore, there is an urgent need to develop a high-purity, high-activity recombinant type III collagen and its preparation method to overcome the above-mentioned technical defects and meet the application needs of the medical and cosmetic fields. Summary of the Invention

[0006] This application addresses the problems in existing technologies, such as insufficient uniqueness in recombinant type III collagen sequence design, low purity of expression strains, and imperfect functional verification systems. It provides a recombinant type III collagen with high purity and high activity, its preparation method, and its applications, which are of key significance in the fields of biomaterials, cosmetics, and high-end medical aesthetics.

[0007] In a first aspect of this application, a recombinant type III collagen is provided, comprising n structural units; said structural unit comprises peptides A, B, and C connected in any order; wherein:

[0008] The A is a peptide with an amino acid sequence as shown in SEQ ID NO:1, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO:1 and derived from human type III collagen.

[0009] The B is a peptide with an amino acid sequence as shown in SEQ ID NO:2, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO:2 and derived from human type III collagen.

[0010] The C is a peptide with an amino acid sequence as shown in SEQ ID NO:3, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO:3 and derived from human type III collagen.

[0011] n is an integer from 1 to 10.

[0012] In a second aspect of this application, a nucleic acid molecule encoding recombinant type III collagen as described in the first aspect is provided.

[0013] In a third aspect of this application, a recombinant expression vector is provided, comprising the nucleic acid molecule as described in the second aspect.

[0014] In a fourth aspect of this application, a recombinant host cell is provided that expresses recombinant type III collagen as described in the first aspect.

[0015] In a fifth aspect of this application, a method for preparing the recombinant type III collagen described in the first aspect is provided, comprising the following steps:

[0016] The recombinant host cells as described in the fourth aspect are cultured under conditions suitable for the expression of the recombinant type III collagen, and the expression of the recombinant type III collagen is induced to prepare a culture; and,

[0017] The recombinant type III collagen was recovered and purified from the culture.

[0018] In a sixth aspect of this application, the recombinant type III collagen as described in the first aspect, the formulation as described in the second aspect, the nucleic acid molecule as described in the third aspect, the recombinant expression vector as described in the fourth aspect, or the recombinant host cell as described in the fifth aspect are provided for use in at least one of the following aspects:

[0019] (1) Application in the preparation of skin repair preparations, tissue regeneration materials, wound dressings, hydrogels or facial fillers;

[0020] (2) Application in the preparation of medical devices or biomedical materials;

[0021] (3) Application in the preparation of cosmetic active ingredients, antioxidant products or anti-inflammatory products.

[0022] In a seventh aspect of this application, a formulation comprising recombinant type III collagen as described in the first aspect is provided. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0024] Figure 1 This is an SDS-PAGE electrophoresis image of the supernatant of recombinant type III collagen (ABC)n fermentation expression in one embodiment (Example 2) of this application.

[0025] Figure 2 This is a high-performance liquid chromatogram of the purified collagen in one embodiment (Example 3) of this application.

[0026] Figure 3 This is a schematic diagram of the circular dichroism spectral verification results of recombinant type III collagen in one embodiment (Example 3) of this application.

[0027] Figure 4 This is a diagram showing the cell scratch results in one embodiment (Example 6) of this application.

[0028] Figure 5 This is a bar graph showing the anti-wrinkle evaluation of the caudal fin area of ​​zebrafish in one embodiment (Example 7) of this application.

[0029] Figure 6This is an SDS-PAGE electrophoresis image of the fermentation expression supernatant of recombinant type III collagen (single repeat) in Comparative Example 1.

[0030] Figure 7 This is an SDS-PAGE electrophoresis image of the supernatant of recombinant type III collagen (BAC)n fermentation expression in one embodiment (Example 8) of this application.

[0031] Figure 8 This is a line graph showing the cytotoxicity results in one embodiment of this application (Comparative Example 1). Detailed Implementation

[0032] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0035] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0036] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0037] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate a connection in the coverage of different technical solutions presented earlier and later, but should not be construed as limiting the scope of the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0038] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0039] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0040] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0041] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0042] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0043] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0044] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately, concurrently, or in turn with other steps or parts of the sub-steps or stages of other steps.

[0045] The "homology" (sequence identity percentage (%)) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment, and, where necessary, the introduction of vacancies to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence by the total number of amino acid residues (or bases) in the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools can be used, such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., *Bioinformatics* (Cambridge, UK), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software can be used to perform alignments to determine the percentage of identity between amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters as needed for the alignment, for example by selecting a suitable algorithm.

[0046] As used in this application, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups, as well as the side chain characteristic of each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, summarized in Table A below.

[0047] Table A

[0048]

[0049] One aspect of this application provides a recombinant type III collagen having a triple helix structure.

[0050] In some embodiments, the recombinant type III collagen comprises n structural units; each structural unit comprises peptides A, B, and C connected in any sequential order; wherein:

[0051] The A is a peptide with an amino acid sequence as shown in SEQ ID NO: 1, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 1 and derived from human type III collagen.

[0052] The B is a peptide with an amino acid sequence as shown in SEQ ID NO: 2, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 2 and derived from human type III collagen.

[0053] The C is a peptide with an amino acid sequence as shown in SEQ ID NO: 3, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 3 and derived from human type III collagen.

[0054] n is an integer from 1 to 10 (inclusive).

[0055] For example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0056] Among them, SEQ ID NO: 1 is shown below:

[0057] GHPGSPGSPGYQGPPGEPGPSGPP;

[0058] SEQ ID NO: 2 is shown below:

[0059] GAPGERGPPGLAGAPGLR;

[0060] SEQ ID NO: 3 is shown below:

[0061] GPAGPRGPVGPSGPPGKD.

[0062] For example, the connection order of A, B and C from N end to C end in the structural unit is: ABC, ACB, BCA, BAC, CAB or CBA.

[0063] In some embodiments, the connection order of A, B, and C in the structural unit from the N end to the C end is ABC.

[0064] In some embodiments, the recombinant type III collagen includes the structure (ABC)n, where n is an integer from 3 to 8, for example, 7.

[0065] In some embodiments, the amino acid sequence of A is as shown in SEQ ID NO: 1.

[0066] In some embodiments, the amino acid sequence of B is as shown in SEQ ID NO: 2.

[0067] In some embodiments, the amino acid sequence of C is as shown in SEQ ID NO: 3.

[0068] In some implementations, A, B, and C are connected in series in the order ABC, and n is 7.

[0069] In some embodiments, the recombinant type III collagen has an amino acid sequence that is at least 85% (e.g., 90% or 95%) identical to the amino acid sequence shown in SEQ ID NO: 4, and has a triple helix structure.

[0070] For example, the sequence of the recombinant type III collagen is shown in SEQ ID NO: 4.

[0071] SEQ ID NO: 4:

[0072] GHPGSPGSPGYQGPPGEPGPSGPPGAPGERGPPGLAGAPGLRGPAGPRGPVGPSGPPGKDGHPGSPGSPGYQGPPGEPGPSGPPGAPGERGPPGLAGAPGLRGPAGPRGPVGPSGPPGKDGHPGSPGSPGYQGPPGEPGPSGPPGAPGERGPPGLAGAPGLRRGPAGPRGPVGPSGPPGKDGHPGSPGSPGYQGPPGEPGPSGPPGAPGER GPPGLAGAPGLRGPAGPRGPVGPSGPPGKDGHPGSPGSPGYQGPPGEPGPSGPPGAPGERGPPGLAGAPGLRGPAGPAGPRGPVGPSGPPGKDGHPGSPGSPGYQGPP GEPGPSGPPGAPGERGPPGLAGAPGLRGPAGPRGPVGPSGPPGKDGHPGSPGSPGYQGPPGEPGPSGPPGAPGERGPPGLAGAPGLRGPAGPRGPVGPSGPPGKD.

[0073] For example, the sequence of the recombinant type III collagen is shown in SEQ ID NO: 7.

[0074] SEQ ID NO: 7:

[0075] GHPGSPGSPGYQGPPGEPGPSGAPGERGPPGLAGAPGPAGPRGPVGPSGPPGHPGSPGSPGYQGPPGPGPSGAPGERGPPGLAGAPGPAGPRGPVGPSGPPGHPGSPGSPGYQGPPGEPGPSGAPGERGPPGLAGAPGPAGPRGPVGPSGPPGHPGSPGSPGYQGPPGEPGPSGAPGE RGPPGLAGAPGPAGPRGPVGPSGPPGHPGSPGSPGYQGPPGPSGAPGERGPPGLAGAPGPAGPRGPVGPSGPPGHPGSPGSPGYQGPPGPSGAPGERGPPGLAGAPGPAGPRGPVGPSGPPGHPGSPGSPGYQGPPGEPGPSGAPGERGPPGLAGAPGPAGPRGPVGPSGPP.

[0076] In some implementations, A, B, and C are connected in series in the order BAC, where n is 7.

[0077] In some embodiments, the recombinant type III collagen has an amino acid sequence that is at least 85% (e.g., 90% or 95%) identical to the amino acid sequence shown in SEQ ID NO: 6, and has a triple helix structure.

[0078] For example, the sequence of the recombinant type III collagen is shown in SEQ ID NO: 6.

[0079] SEQ ID NO: 6:

[0080] GAPGERGPPGLAGAPGLRGHPGSPGSPGYQGPPGEPGPSGPPGPAGPRGPVGPSGPPGKDGAPGERGPPGLAGAPGLRGHPGSPGSPGYQGPPGEPGPSGPPGPAGPRGPVGPSGPPGKDGAPGERGPPGLAGAPGLRGHPGSPGSPGYQGPPGEPGPSGPPGPAGPRGPVGPSGPPGKDGAPGERGPPGLAGAPGLRGHPGSPGSPGYQ GPPGEPGPSGPPGPAGPRGPVGPSGPPGKDGAPGERGPPGLAGAPGLRGHPGSPGSPGYQGPPGEPGPSGPPGPAGPRGPVGPSGPPGKDGAPGERGPPGLAGAP GLRGHPGSPGSPGYQGPPGEPGPSGPPGPAGPRGPVGPSGPPGKDGAPGERGPPGLAGAPGLRGHPGSPGSPGYQGPPGEPGPSGPPGPAGPRGPVGPSGPPGKD.

[0081] Another aspect of this application provides a protein construct comprising recombinant type III collagen as described above.

[0082] In some implementations, the protein construct further includes an affinity tag. Examples include, for instance, a Flag tag, a His tag, a Strep-tag II tag, or an Avi-tag.

[0083] Another aspect of this application provides a nucleic acid molecule that encodes the aforementioned recombinant type III collagen containing a triple helix structure.

[0084] In this application, unless otherwise stated, "nucleic acid" has the well-known meaning in the art and is also called "polynucleotide," which is a molecule formed by multiple nucleotide monomers.

[0085] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5.

[0086] SEQ ID NO: 5:

[0087]

[0088] In another aspect of this application, a formulation comprising the recombinant type III collagen as described above is provided.

[0089] In this application, the formulations include, but are not limited to, skin repair formulations, tissue regeneration materials, cosmetics, pharmaceuticals, and protein formulations.

[0090] In some embodiments, the purity of the recombinant type III collagen in the protein formulation is greater than or equal to 90%; exemplaryly, greater than or equal to 95%; or greater than or equal to 99%.

[0091] In another aspect of this application, a recombinant expression vector is provided, which includes the nucleic acid molecules described above.

[0092] Unless otherwise specified, the term "vector" in this application refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element. Vectors can be, for example, plasmids, granules, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), RNA vectors, or linear or circular DNA or RNA molecules, which may include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. This term includes vectors that serve as self-replicating nucleic acid structures as well as vectors incorporated into the host cell genome. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked.

[0093] In some embodiments, the backbone vector of the recombinant expression vector includes one or more of the pPIC9K, pPICZaA, and pET series. For example, the backbone vector of the recombinant expression vector is pPIC9K.

[0094] In another aspect of this application, a recombinant host cell is provided that expresses the recombinant type III collagen as described above.

[0095] In some embodiments, the recombinant host cell comprises the nucleic acid molecules described above and / or the recombinant expression vector described above; exemplaryly, the nucleic acid molecules described above are integrated into the genome.

[0096] In some embodiments, the chassis cells of the recombinant host cell are eukaryotic cells or prokaryotic cells.

[0097] For example, the prokaryotic cells are Escherichia coli or Bacillus.

[0098] In some embodiments, the eukaryotic cells include Pichia pastoris.

[0099] In some embodiments, the protease B (also referred to as "PR1 protease" or "PRB1 protease") gene and / or protease A (also referred to as "PEP4 protease") gene of the recombinant host cell are knocked out or inactivated; exemplaryly, the recombinant host cell knocks out the PRB1 and PEP4 protease genes using CRISPR-Cas9 technology.

[0100] Another aspect of this application provides a method for preparing recombinant type III collagen as described above.

[0101] In some embodiments, the method includes:

[0102] The recombinant host cells described above are cultured under conditions suitable for the expression of the recombinant type III collagen to induce the expression of the recombinant type III collagen, and a culture is prepared; and,

[0103] The recombinant type III collagen was recovered and purified from the culture.

[0104] In some implementations, the method includes the following steps:

[0105] (1) Construct a recombinant expression vector containing a nucleotide sequence encoding the recombinant type III collagen;

[0106] (2) Transform the recombinant expression vector from step (1) into host cells to prepare recombinant host cells;

[0107] (3) The recombinant host cells are cultured under suitable expression conditions, and the expression of the recombinant type III collagen is induced to obtain a culture;

[0108] (4) The recombinant type III collagen was recovered and purified from the culture.

[0109] In one specific embodiment of this application, the host cell in step (2) is Pichia pastoris, and its PR1 and PEP4 protease genes are knocked out by CRISPR-Cas9 technology.

[0110] In one specific embodiment of this application, the culture conditions in step (3) include a temperature of 25~32℃, a pH of 5.5~6.8, and methanol-induced expression.

[0111] In one specific embodiment of this application, the dissolved oxygen during the cultivation process in step (3) is 20%~30%.

[0112] In one specific embodiment of this application, the induction temperature is 26°C to 30°C, and the pH is 4.5 to 5.5. The induction time can be 48 h to 120 h, for example, 114 h.

[0113] In one specific embodiment of this application, the purification in step (4) includes ultrafiltration and ion exchange chromatography.

[0114] In one specific embodiment of this application, the host cell in step (2) is Escherichia coli.

[0115] In one specific embodiment of this application, the culture conditions in step (3) include a temperature of 16℃~37℃, a rotation speed of 150 rpm~220 rpm, and IPTG-induced expression. Exemplarily, the induction time is 12 h~16 h.

[0116] In another aspect of this application, the use of the recombinant type III collagen as described above, the nucleic acid molecule as described above, the recombinant expression vector as described above, or the recombinant host cell as described above is also provided.

[0117] The applications include one or more of the following:

[0118] (1) Application in the preparation of skin repair agents, tissue regeneration materials, wound dressings, hydrogels, and facial fillers;

[0119] (2) Applications in the preparation of medical devices and biomedical materials;

[0120] (3) Application in the preparation of cosmetic active ingredients, antioxidant products or anti-inflammatory products.

[0121] In one specific embodiment of this application, the recombinant type III collagen is used in the form of solution, dry powder, or gel; for example, the dry powder form is a sponge-like freeze-dried product.

[0122] In some embodiments, the medical device includes any one or more of recombinant type III collagen hydrogel, wound dressing, facial filler, and tissue engineering material.

[0123] In the above applications, recombinant type III collagen can exist in solution, dry powder, or gel form.

[0124] One or more of the above technical solutions have the following advantages or beneficial effects:

[0125] (1) The three specific sequences (A, B, C) of the selected human type III collagen are tandemly repeated in the order of ABC or BAC, which can form a highly bioactive recombinant type III collagen with a triple helix structure, which is stable in expression and easy to produce on a large scale.

[0126] (2) By example, the PR1 and PEP4 protease genes of Pichia pastoris were knocked out by CRISPR-Cas9 technology to construct an anti-degradation engineered strain, making the recombinant type III collagen less prone to degradation during fermentation, and the fermentation purity can reach more than 90%, which greatly reduces the production cost.

[0127] (3) The recombinant type III collagen produced has excellent properties such as promoting cell proliferation, cell adhesion, cell migration and anti-oxidation, and has extremely high application value in the fields of skin repair and tissue regeneration.

[0128] (4) The amino acid sequence of this recombinant type III collagen is 100% homologous to the corresponding fragment of natural human type III collagen. It has low immunogenicity and can be safely applied in the fields of medical devices, cosmetics and biomedicine.

[0129] The following are some examples.

[0130] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0131] The YPG medium used in the following examples is a conventional culture medium formulation (10 g / L yeast extract, 20 g / L peptone and 30 g / L glycerol).

[0132] Example 1: Gene Design and Expression Vector Scheme

[0133] 1. Gene design

[0134] This embodiment provides a recombinant yeast strain expressing recombinant type III collagen with the amino acid sequence shown in SEQ ID NO: 4 and its construction process. The recombinant type III collagen is formed by arranging three amino acid unit sequences (A, B, C) in the form of (ABC)n, repeated 7 times. The amino acid sequences of its monomer units (repeated once) are shown in SEQ ID NO: 1 (A), SEQ ID NO: 2 (B), and SEQ ID NO: 3 (C), and the full-length amino acid sequence after 7 repetitions is shown in SEQ ID NO: 4.

[0135] In one typical embodiment, this application provides a nucleic acid molecule encoding the aforementioned recombinant type III collagen. The nucleotide unit sequence of the recombinant collagen fragment is shown in SEQ ID NO: 5.

[0136] 2. Construction of expression vector

[0137] The optimized nucleotide sequence SEQ ID NO: 5 was synthesized into a whole gene and ligated into the expression vector pPIC9K. The correctness of the inserted fragment was verified by sequencing, thus obtaining the pPIC9K-COL1 recombinant plasmid. The recombinant plasmid was then transformed into Escherichia coli DH5α competent cells by ice bath and heat shock to obtain the DH5α / pPIC9K-COL1 recombinant Escherichia coli strain containing the plasmid.

[0138] Example 2: Fermentation expression of recombinant type III collagen

[0139] This embodiment provides a method for constructing and expressing bacterial strains using the expression vector in Example 1.

[0140] 1) Construction of anti-degradation engineered strains

[0141] Using Pichia pastoris strain GS115 as the starting strain, sgRNAs targeting the PRB1 and PEP4 protease genes were designed. The two protease genes were then knocked out at specific sites using the CRISPR-Cas9 system to eliminate the degradation of the target protein by the host protease. The knockout efficiency and accuracy were verified by PCR. Strains with missing PR1 and PEP4 protease gene fragments were screened to obtain the degradation-resistant engineered strain GS115B-ΔPR1-ΔPEP4 (hereinafter referred to as GS115B).

[0142] 2) Construction of recombinant type III collagen strains

[0143] Using the aforementioned anti-degradation engineered strain GS115B as the expression host, the pPIC9K-COL1 plasmid fragment digested with restriction endonuclease Sal I was transformed into the anti-degradation engineered strain GS115B by electroporation. Transformants were obtained by culturing at 30℃ for 48 hours. Sequencing confirmed successful gene integration, and the GS115B / pPIC9K-COL1 recombinant type III collagen expression strain was obtained.

[0144] 3) Achieving the induced expression of recombinant type III collagen in Pichia pastoris.

[0145] After activating the GS115B / pPIC9K-COL1 strain by streak plating, single colonies were picked and seed cultured in YPG medium until OD500 was reached. 600The first-stage seed culture was obtained at 13:00 and inoculated into a fermenter with an initial volume of 2.5 L at a 10% inoculation rate. The culture temperature was 30℃, pH 5.5, and dissolved oxygen was controlled at 20%~30%. After the glycerol was exhausted, glycerol feeding culture was started. When the cell wet weight reached more than 100 g / L, methanol was switched to induce culture. The induction stage temperature was 28℃ and pH 5.0. After 114 h of induction, the fermenter was removed from the tank, and the supernatant was collected by centrifugation. The crude protein content of the fermentation supernatant was found to be 6.2 g / L and the purity was greater than 85% by high performance liquid chromatography (HPLC).

[0146] 4) Achieve induced expression of recombinant type III collagen in Escherichia coli.

[0147] The cultivation conditions are as follows:

[0148] 1) Construct the COL1 fragment into the pET28a plasmid. Successful construction yields the pET28a-COL1 recombinant plasmid.

[0149] 2) Transform the above plasmid into BL21(DH5α) Escherichia coli. After overnight culture, randomly select several single colonies and inoculate them into 10 mL of LB medium containing 100 mg / mL ampicillin. After culturing for 4-5 hours until the bacterial culture becomes turbid, inoculate them into 500 mL of LB medium containing an appropriate amount of 100 mg / mL ampicillin. Incubate at 37°C with shaking at 220 rpm until the culture reaches the OD value. 600 The concentration of the solution was 0.4-0.6. IPTG was added to induce fermentation to a final concentration of 0.4 mM. Fermentation was stopped after 12-16 h, the supernatant was discarded by centrifugation, and the cells were collected.

[0150] 3) Resuspend the bacterial cells in ultrasonic lysis buffer (formulation: 20 mM Tris-HCl, 400 mM NaCl, 1 mM PMSF, pH 8.0) at a ratio of 1 g: 5 mL wet weight of bacterial cells to buffer volume. Then, sonicate the bacterial suspension in an ice-water bath. After lysis, separate the supernatant and precipitate. Filter the supernatant through a 0.45 μm filter membrane. Protein electrophoresis results showed successful expression of recombinant type III collagen.

[0151] Example 3: Purification of recombinant type III collagen

[0152] The fermentation supernatant from the induced expression in Example 2 was collected by centrifugation.

[0153] (1) Ion exchange chromatography: The ultrafiltration concentrate was loaded onto a HiTrap Q HP anion exchange chromatography column that had been pre-equilibrated with 20 mM PBS buffer (pH 7.4). After loading, unbound proteins were washed away with equilibration buffer, and then linear gradient elution was performed with PBS buffer containing 0-1 M NaCl. The target elution peak was collected.

[0154] (2) Ultrafiltration: The supernatant was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and washed with 20 mM PBS buffer (pH 7.4) to replace the buffer and remove some small molecule impurities.

[0155] (3) The purity of the target protein was analyzed by high performance liquid chromatography (HPLC). The chromatographic column was a TSK gel G3000SWXL gel filter column (7.8×300mm), the mobile phase was 0.1M PB + 0.1M Na2SO4 (pH 7.0), the flow rate was 0.5mL / min, the detection wavelength was 220nm, the column temperature was 28℃, and the loading volume was 30μL. The results calculated by the area normalization method showed that the percentage of the main peak area of ​​the target protein was 97%, indicating that its purity was higher than 95%.

[0156] (4) Final processing: The collected target protein eluent was concentrated by ultrafiltration, sterilized and filtered, packaged and freeze-dried to obtain recombinant type III collagen sponge-like freeze-dried product.

[0157] Example 4: Circular dichroism spectroscopy verification of recombinant type III collagen

[0158] The lyophilized sponge prepared in Example 3 was reconstituted with PBS solution and subjected to circular dichroism spectroscopy. The starting wavelength was set to 190 nm, the ending wavelength to 250 nm, the step size to 1 nm, and the experiment was repeated three times. The acquisition time was 0.5 s / point, and the cuvette width was 0.1 cm. The results are as follows: Figure 6 As shown, collagen has a negative peak at 198nm and a positive peak at 221nm, which is consistent with the CD characteristics of triple-helix collagen.

[0159] Example 5: Evaluation of the cytotoxicity of recombinant type III collagen

[0160] (1) Experimental materials:

[0161] Dermal fibroblasts, mouse mononuclear macrophages, and rat basophilic leukemia cells; cell count per group: 5 × 10⁻⁶ 3 One hole / hole

[0162] The experimental group was selected from the recombinant type III collagen sample purified in Example 3;

[0163] The control group includes a blank control group and a normal control group;

[0164] (2) Experimental steps

[0165] Dermal fibroblasts, mouse mononuclear macrophages, and rat basophilic leukemia cells were inoculated into 96-well plates (5 × 10⁻⁶ cells / well). 3 (each well contains 1 cell / well) and incubated at 37 °C in 5% CO2 for 24 h. The groups are shown in Table 1.

[0166] Table 1

[0167]

[0168] Drug administration: Fresh culture medium containing the corresponding concentration of the sample was added to the sample group, while the normal control group and blank control group were replaced with fresh culture medium and incubated at 37 ℃ and 5% CO2 for 24 h.

[0169] After incubation, add MTT solution to each well and continue culturing for 4 h.

[0170] Remove the culture medium, add DMSO solution, shake to mix, and measure the absorbance at 490 nm.

[0171] .

[0172] Statistical results are expressed as mean ± SD and statistical analysis was performed using SPSS software. A p < 0.05 (compared to the normal control group) indicated a significant difference, and cell viability less than 95% was considered toxic.

[0173] (3) Result evaluation

[0174] As shown in Table 2, recombinant type III collagen showed no cytotoxicity in human dermal fibroblasts at a concentration range of 0.625% (v / v); and no cytotoxicity in mouse mononuclear macrophages and rat basophilic leukemia cells at a concentration range of 0.312% (v / v). It also exhibited a certain proliferative effect at the indicated concentration ranges.

[0175] Table 2 Cytotoxicity Evaluation

[0176]

[0177] Example 6: Evaluation of the cell scratch / repair efficacy of recombinant type III collagen

[0178] (1) Experimental materials:

[0179] Mouse embryonic fibroblasts (NIH / 3T3)

[0180] The experimental group was selected from the recombinant type III collagen sample purified in Example 3;

[0181] The control group includes a blank control group and a normal control group;

[0182] (2) Experimental steps

[0183] Cells were seeded in 12-well plates and incubated at 37 °C with 5% CO2 for 24 h. The experimental groups are shown in Table 3.

[0184] Table 3

[0185]

[0186] Scratches were created and washed twice with PBS (gentle). The normal control group was treated with fresh culture medium, while the sample groups were treated with fresh culture medium containing the corresponding concentration of the sample. Three biological replicates were performed, and the samples were incubated at 37 ℃ and 5% CO2 for 24 ± 2 h. Images were taken at 0 h and 24 h after drug administration, and the scratch area was recorded. Image J analysis was used to analyze the scratch area in each group, and the healing area (Y) was calculated. Statistical analysis showed p < 0.05 as a significant difference.

[0187] .

[0188] (3) Result evaluation

[0189] This implementation example Figure 4 As shown, the scratch healing area of ​​the collagen sample group was 499±31.4, while the healing area of ​​the normal control group was 409±8.72, which was significantly increased, with an improvement rate of 22%, revealing that the collagen sample has a repairing effect.

[0190] Example 7: Evaluation of anti-wrinkle / repair efficacy using a zebrafish model

[0191] (1) Experimental materials:

[0192] Experimental system: Wild-type AB strain zebrafish. The zebrafish were 2 days post-fertilization (2 dpf). The adult fish rearing and breeding methods met the requirements of the international AAALAC certification (certification number: 001458).

[0193] The experimental group was selected from the recombinant type III collagen sample purified in Example 3;

[0194] The control group includes a normal control group and a model control group;

[0195] (2) Experimental steps

[0196] Evaluation of anti-wrinkle efficacy: Zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. Water-soluble samples were administered, with a normal control group and a model control group included. Each well contained 3 mL of water. After incubation at 28 ℃ in the dark for 2 h, except for the normal control group, zebrafish in all experimental groups were irradiated under ultraviolet light three times, each irradiation lasting 15 min, with a 30 min interval. They were then incubated at 28 ℃ in the dark for another 22 h. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data. The caudal fin area (A) of the zebrafish was analyzed, and the anti-wrinkle efficacy of the samples was calculated and determined according to the formula (see Table 4).

[0197] .

[0198] Statistical analysis showed p < 0.05, which was considered a significant difference.

[0199] Table 4

[0200]

[0201] (3) Efficacy evaluation

[0202] The tail fin area of ​​the recombinant type III collagen group was significantly increased compared with that of the model control group, revealing that this sample has a good anti-wrinkle effect (see...). Figure 5 ).

[0203] In summary, by selecting appropriate amino acid sequences, this application can obtain recombinant type III collagen with superior biological activity. The selection of more suitable repeat combinations can improve the effects of recombinant collagen products on cell proliferation, repair, and anti-wrinkle efficacy, making them suitable for medical devices or medical products such as tissue repair, anti-oxidation, and excipients.

[0204] Example 8

[0205] This embodiment provides a recombinant yeast strain expressing recombinant type III collagen with the amino acid sequence shown in SEQ ID NO: 6 and its construction process. The recombinant type III collagen is formed by arranging three amino acid unit sequences (A, B, C) in the form of (BAC)n, repeated 7 times.

[0206] The fermentation expression and purification methods for recombinant type III collagen strains are the same as those in Examples 2 and 3.

[0207] In this embodiment, the recombinant type III collagen began to show significant degradation after 78 hours of fermentation, and its purity was lower than that of Example 3. However, the target protein was still visible (see Example 3). Figure 7 ).

[0208] Example 9

[0209] This embodiment provides one or more recombinant type III collagen variants. While keeping the core functional regions of the peptides unchanged, the effect of shortening the terminal amino acids of each peptide unit on collagen activity was examined.

[0210] The specific sequence changes are as follows: three amino acids are deleted from the ends of peptides A, B, and C, respectively. These are then repeated seven times in the (ABC)n format to form the collagen variant. The amino acid sequence is shown in SEQ ID NO: 7. This sequence shows 85% homology to the sequence shown in SEQ ID NO: 4.

[0211] The fermentation expression and purification methods for the recombinant type III collagen variant strain were the same as those in Examples 2 and 3.

[0212] The cytotoxicity evaluation of the recombinant type III collagen variant was consistent with that in Example 5.

[0213] As shown in Table 5, shortening the lengths on both sides of the amino acid unit sequence did not alter the core collagen function. The collagen variant samples showed no cytotoxicity at higher concentrations and exhibited a certain proliferative effect at the indicated concentration range. These results are equivalent to or better than those in Example 5.

[0214] Table 5 Toxicity evaluation of recombinant type III collagen variants

[0215]

[0216] Comparative Example 1

[0217] In this comparative example, the recombinant type III collagen was formed by repeating the amino acid sequence SEQ ID NO: 1 1-10 times. The fermentation expression and purification methods of the recombinant type III collagen strain were the same as those in Examples 2 and 3.

[0218] Experimental results showed that recombinant collagen III could be expressed after repeating the amino acid sequence SEQ ID NO: 1 3-10 times (see [link]). Figure 6 However, due to the high repetitiveness of its sequence, the recombinant protein in this comparative example not only failed to self-assemble into a stable triple helix structure, but also, in a toxicity experiment on human dermal fibroblasts, the recombinant type III collagen formed by selecting an amino acid sequence repeated 10 times showed a significant inhibitory effect on cell growth. Compared with the blank control group, the cell viability of the treatment group in Comparative Example 1 was significantly reduced (refer to...). Figure 8 At a dosage concentration of 0.039%, the recombinant type III collagen in this comparative example exhibited significant cytotoxicity against human dermal fibroblasts, which was far less than the dosage concentrations of the recombinant type III collagen in Examples 1-9. This indicates that the recombinant type III collagen in this comparative example is not suitable for practical applications.

[0219] The toxicity test on human dermal fibroblasts was the same as in Example 5.

[0220] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0221] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Recombinant type III collagen, characterized in that, It comprises n structural units; each structural unit includes peptides A, B, and C connected in any order; wherein: The A is a peptide with an amino acid sequence as shown in SEQ ID NO: 1, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 1 and derived from human type III collagen. The B is a peptide with an amino acid sequence as shown in SEQ ID NO: 2, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 2 and derived from human type III collagen. The C is a peptide with an amino acid sequence as shown in SEQ ID NO: 3, or a functional variant peptide with at least 85% identity to the amino acid sequence shown in SEQ ID NO: 3 and derived from human type III collagen. n is an integer from 1 to 10.

2. The recombinant type III collagen as described in claim 1, characterized in that, The recombinant type III collagen includes the structure (ABC)n or (BAC)n, where n is an integer from 3 to 8.

3. The recombinant type III collagen as described in claim 1 or 2, characterized in that, It satisfies one or more of the following conditions: (1) The amino acid sequence of A is shown in SEQ ID NO: 1; (2) The amino acid sequence of B is shown in SEQ ID NO: 2; (3) The amino acid sequence of C is shown in SEQ ID NO: 3; (4) n is 7.

4. The recombinant type III collagen as described in claim 3, characterized in that, Its amino acid sequence has at least 85% identity with the amino acid sequences shown in SEQ ID NO:4 or SEQ ID NO:6, and it has a triple helix structure; Optionally, the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:

7.

5. A formulation comprising recombinant type III collagen as described in any one of claims 1 to 4; Optionally, the formulation includes one or more of the following: skin repair formulations, tissue regeneration materials, cosmetics, pharmaceuticals, and protein formulations; In the protein formulation, the purity of the recombinant type III collagen is optionally greater than or equal to 90%; further optionally greater than or equal to 95%; and even further optionally greater than or equal to 99%.

6. A nucleic acid molecule encoding recombinant type III collagen as described in any one of claims 1 to 4.

7. A recombinant expression vector, characterized in that, It includes the nucleic acid molecules as described in claim 6.

8. Recombinant host cells, characterized in that, It expresses recombinant type III collagen as described in any one of claims 1 to 4; Optionally, the recombinant host cell comprises the nucleic acid molecule as described in claim 6 and / or the recombinant expression vector as described in claim 7; Optionally, the chassis cells of the recombinant host cell are eukaryotic cells or prokaryotic cells; wherein: The prokaryotic cells may optionally be Escherichia coli or Bacillus; The eukaryotic cells may optionally include Pichia pastoris, in which the genes expressing protease B and / or protease A are knocked out or inactivated.

9. A method for preparing the recombinant type III collagen according to any one of claims 1 to 4, characterized in that, Includes the following steps: Cultures are prepared by culturing the recombinant host cells as described in claim 8 under conditions suitable for the expression of the recombinant type III collagen, inducing the expression of the recombinant type III collagen, and preparing a culture. The recombinant type III collagen was recovered and purified from the culture. Optionally, the recombinant host cell is a Pichia pastoris whose gene expressing protein B and / or protein A has been knocked out or inactivated; the steps for preparing the culture meet one or more of the following conditions: (1) The conditions for culturing the Pichia pastoris include: a temperature of 25℃~32℃, a pH of 5.5~6.8, and a dissolved oxygen content of 20%~30%; (2) Carbon source supplementation during the cultivation process; (3) The conditions for inducing the expression of the recombinant type III collagen include: a temperature of 26℃~30℃ and a pH of 4.5~5.5; (4) The induction was performed using methanol; (5) The induction duration is 48h~120h; Optionally, the recombinant host cell is *Escherichia coli*, and the steps for preparing the culture meet one or more of the following conditions: 1) The conditions for culturing the Escherichia coli include: a temperature of 16℃~37℃ and a rotation speed of 150 rpm~220 rpm; 2) Cultivate to OD 600 IPTG was used for induction culture when the pH was 0.4~0.

6. 3) The induction culture time is 12 h to 16 h.

10. The use of the recombinant type III collagen as described in any one of claims 1 to 4, the formulation as described in claim 5, the nucleic acid molecule as described in claim 6, the recombinant expression vector as described in claim 7, or the recombinant host cell as described in claim 8 in at least one of the following aspects: (1) Application in the preparation of skin repair preparations, tissue regeneration materials, wound dressings, hydrogels or facial fillers; (2) Application in the preparation of medical devices or biomedical materials; (3) Application in the preparation of cosmetic active ingredients, antioxidant products or anti-inflammatory products.

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