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JP-7855803-B2 - Method for biosynthesizing type IV collagen, a structural material for the human body.

JP7855803B2JP 7855803 B2JP7855803 B2JP 7855803B2JP-7855803-B2

Inventors

  • ▲楊▼ 霞
  • ▲劉▼ ▲増▼耀
  • ▲蘭▼ 小▲賓▼
  • 王 玲玲
  • ▲張▼ 永健
  • ▲劉▼ 欣
  • ▲張▼ ▲暁▼波
  • 何 振瑞

Assignees

  • 山西▲錦▼波生物医▲薬▼股▲フン▼有限公司

Dates

Publication Date
20260508
Application Date
20231221
Priority Date
20231025

Claims (19)

  1. Recombinant collagen comprising 2 to 10 directly linked repeating units , wherein each repeating unit consists of an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90 % identity with the amino acid sequence of SEQ ID NO: 1 . Recombinant collagen with cell adhesion activity.
  2. Recombinant collagen according to claim 1, comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90 % identity with the amino acid sequence of SEQ ID NO: 2.
  3. A nucleic acid encoding recombinant collagen as described in claim 1.
  4. The nucleic acid according to claim 3 , comprising the nucleotide sequence of S EQ ID NO: 3.
  5. A vector comprising the nucleic acid described in claim 3.
  6. The vector according to claim 5, comprising an expression control element operably linked to the nucleic acid, a nucleotide sequence of a purified tag, and/or a nucleotide sequence of a leader sequence.
  7. The expression control element is selected from the group consisting of promoters, terminators, or enhancers. The vector according to claim 6, wherein the purified tag is selected from the group consisting of His tag, GST tag, MBP tag, SUMO tag, or NusA tag.
  8. A host cell comprising the nucleic acid described in claim 3.
  9. The host cell according to claim 8, which is a eukaryotic cell or a prokaryotic cell.
  10. The host cell according to claim 9, wherein the eukaryotic cell is a yeast cell, an animal cell and/or an insect cell, and/or the prokaryotic cell is an Escherichia coli cell .
  11. A composition comprising recombinant collagen as described in claim 1 .
  12. The composition according to claim 11, which is one or more of the following: biomimetic materials, human biomimetic materials, cosmetic materials, organoid culture materials , cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biological materials, nerve repair and regeneration materials, liver tissue materials and blood vessel repair and regeneration materials, 3D printed artificial organ biological materials, cosmetic raw materials, medicinal auxiliary materials and food additives .
  13. The composition according to claim 11, which is a surface composition, an injection composition, or an oral composition.
  14. Use of recombinant collagen according to claim 1 or 2, nucleic acid according to claim 3 or 4 , vector according to any one of claims 5 to 7 and/or host cell according to any one of claims 8 to 10 in the manufacture of one or more of the following: biocoating materials, human biomimetic materials, cosmetic materials, organoid culture materials , cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biological materials, nerve repair and regeneration materials, liver tissue materials and blood vessel repair and regeneration materials, 3D printed artificial organ biological materials, cosmetic raw materials, medicinal auxiliary materials and food additives.
  15. An in vitro method for promoting cell adhesion, comprising the step of bringing cells into contact with recombinant collagen according to claim 1 or 2 in vitro .
  16. The in vitro method according to claim 15, wherein the cells are animal cells.
  17. Recombinant collagen according to claim 1 or 2 , for use in a cosmetic method comprising the step of administering the recombinant collagen according to claim 1 or 2 to a subject.
  18. (1) A step of culturing host cells containing a vector having nucleic acid encoding recombinant collagen under appropriate culture conditions, (2) Harvesting host cells and/or culture medium containing recombinant collagen, A method for producing recombinant collagen according to claim 1 or 2, comprising the step (3) of purifying recombinant collagen.
  19. The method according to claim 18, wherein the host cell is an Escherichia coli cell.

Description

Cross-reference of related applications This application claims priority to a Chinese patent application filed on October 25, 2023, with application number 202311391711.1, and titled "Method for Biosynthesis of Human Body Structural Material Type IV Collagen". This invention belongs to the field of biopharmaceutical technology and relates to recombinant type IV humanized collagen, its production method, and its uses. Collagen (COL), abbreviated as "凝原" in Chinese, is a functional protein in the form of a helical fiber composed of three peptide chains. It is a major component of the extracellular matrix, abundant in the body, and has a wide coverage. In the human body, collagen accounts for 25% to 30% of total protein content and is mainly found in the skin, tendons, and bones. It plays a crucial role in protecting and connecting tissues, and exerts important physiological functions within the body. Type IV collagen, along with laminin, is a major protein that constitutes the basement membrane. Its constituent unit is a trimer with a helical structure composed of three α-chains. While the subunits constituting the trimer differ depending on the tissue, in many tissues, such as the liver, the trimer consists of two α1 chains and one α2 chain. Type IV collagen molecules have structures unique to type IV collagen molecules at both ends of the TH domain that forms the helical structure; the N-terminal structure is called the 7S domain, and the C-terminal structure is called the NC1 domain. Collagen possesses excellent biocompatibility, biodegradability, and low antigenicity. Due to its unique biological structure, it has become a focus of recent research and is widely applied in many fields, including biomedicine, cosmetics, health foods, and food products. The human body contains 28 different types of collagen, which can be classified into two types based on whether their structure is fibrous or not: fibrous and non-fibrous. Fibrous collagen primarily functions as a cell scaffold, fixing cell position, exhibiting anchoring action, and providing tensile strength and rigidity to tissues. Non-fibrous collagen is further subdivided into basal collagen, short-chain collagen, transmembrane collagen, etc., each performing different functions. Conventional collagen production methods involve purifying animal-derived tissues using acid, alkali, and enzymatic hydrolysis to extract collagen derivatives. However, collagen obtained by these methods has already lost its original biological activity, has poor water solubility, does not readily bind to the human body, carries the risk of viral infection and sensitization, and cannot exhibit its true function. Some research institutions express human-derived collagen in vitro using conventional recombinant expression methods, but this is costly, has a long production cycle, and cannot be performed on a large scale. Therefore, there is an urgent need in the market for collagen materials that possess excellent biomaterial properties, have an amino acid sequence highly homologous to that of the human body, and can be mass-produced in industrial systems. The inventors conducted a large-scale functional domain screening of human type IV collagen and discovered 11 recombinant collagens. These recombinant collagens can be expressed in E. coli and purified. Furthermore, the inventors found that these recombinant collagens exhibit high yield, good purity of the target protein after precise purification, and higher activity than the positive control (bovine type I collagen) in cell adhesion activity measurements. In one embodiment, the present invention provides recombinant collagen comprising one or more repeating units, the repeating units being linked directly or via linkers, and the repeating units comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (Gakgdkgskgevgfpglagspgipgskgeq) or 28 (Gptgpagqkgepgssdgipgsagekgepglp) or a variant thereof, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence. In one embodiment, the number of repeating units is 2 to 50, for example, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 4 to 10, or 6 to 10. For example, the number of repeating units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, or a range between these. In one embodiment, the linker contains one or more amino acid residues, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues. In one embodiment, the mutation is selected from substitution, addition, insertion, or deletion. In one embodiment, the substitution is a conservative amino acid substitution. In one embodiment, recombinant collagen is recombinant human type IV collagen