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CN-120842366-B - Polypeptide, recombinant XIX type humanized collagen and application thereof

CN120842366BCN 120842366 BCN120842366 BCN 120842366BCN-120842366-B

Abstract

The invention relates to the technical field of biology, and particularly discloses a polypeptide, recombinant XIX type humanized collagen and application thereof. The amino acid sequence of the polypeptide comprises n repeated units, wherein the repeated units comprise any one of the amino acid sequences shown in any one of SEQ ID NO.1-2 or variant sequences thereof, (2) the amino acid sequences which have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences shown in any one of SEQ ID NO.1-2, and (3) the variant sequences obtained by mutating one or more amino acid residues of the amino acid sequences shown in any one of SEQ ID NO.1-2, wherein n is more than or equal to 1 and is an integer. The polypeptide has a collagen triple helix structure and good cell adhesion activity, can perform the function of human collagen, has no immunological rejection and anaphylactic reaction, and can be used in various fields of cartilage repair, biological dressing, tissue filling and the like.

Inventors

  • YANG XIA
  • LIU XIN
  • ZHANG XIAOBO
  • YANG JINGBO
  • ZHANG YONGJIAN
  • WANG JIAN
  • HE ZHENRUI

Assignees

  • 山西锦波生物医药股份有限公司

Dates

Publication Date
20260508
Application Date
20250530

Claims (20)

  1. 1. A polypeptide, characterized in that the amino acid sequence of the polypeptide consists of n repeat units; the amino acid sequence of the repeating unit is shown as SEQ ID NO.1, wherein n is 5.
  2. 2. The polypeptide of claim 1, wherein the repeating units are directly linked, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 3.
  3. 3. A recombinant XIX-type humanized collagen, characterized in that the recombinant XIX-type humanized collagen comprises the polypeptide of any one of claims 1-2.
  4. 4. The recombinant XIX-type humanized collagen according to claim 3, wherein the recombinant XIX-type humanized collagen has cell adhesion activity.
  5. 5. A polynucleotide encoding the polypeptide of any one of claims 1-2 or the recombinant XIX-type humanized collagen of claim 3 or 4.
  6. 6. The polynucleotide according to claim 5, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID No.5 or a degenerate sequence thereof.
  7. 7. The polynucleotide of claim 5, wherein the polynucleotide further comprises a sequence encoding a purification tag and/or a leader sequence.
  8. 8. The polynucleotide of claim 7, wherein the purification tag is selected from the group consisting of a His tag, a GST tag, an MBP tag, a SUMO tag, and a NusA tag.
  9. 9. A biological material, characterized in that it comprises the polynucleotide according to any one of claims 5 to 8, and that it is an expression cassette, a vector or a host cell.
  10. 10. The biomaterial of claim 9, wherein the vector is an expression vector.
  11. 11. The biomaterial of claim 10, wherein the vector further comprises a control element operably linked to the polynucleotide.
  12. 12. The biomaterial of claim 11, wherein the control element is a promoter, terminator and/or enhancer.
  13. 13. The biomaterial of claim 9, wherein the host cell is a bacterial, fungal or animal cell.
  14. 14. The biomaterial of claim 13, wherein the bacteria comprises E.coli and the fungus comprises yeast.
  15. 15. The biomaterial of claim 14, wherein the fungus is saccharomyces cerevisiae or pichia pastoris.
  16. 16. The method for producing a polypeptide according to any one of claims 1 to 2 or a recombinant XIX-type humanized collagen according to claim 3 or 4, comprising culturing a host cell comprising the polynucleotide according to any one of claims 5 to 8, harvesting the host cell and/or the culture broth comprising the polypeptide or the recombinant XIX-type humanized collagen, and isolating the polypeptide or the recombinant XIX-type humanized collagen therefrom.
  17. 17. A composition for promoting cell adhesion, comprising one or more polypeptides according to any one of claims 1 to 2, and recombinant XIX-type humanized collagen according to claim 3 or 4.
  18. 18. The composition of claim 17, wherein the composition is a topical composition.
  19. 19. The composition of claim 17, wherein the composition is in the form of a solution, a lyophilized powder, a gel, a sponge, or a fiber.
  20. 20. Use of the polypeptide of any one of claims 1-2, the recombinant XIX-type humanized collagen of claim 3 or 4, the polynucleotide of any one of claims 5-8, the biomaterial of any one of claims 9-15, or the composition of any one of claims 17-19 in the preparation of a product for any one or more of cell adhesion, tissue repair, tissue filling or compatibilization.

Description

Polypeptide, recombinant XIX type humanized collagen and application thereof Technical Field The invention relates to the technical field of biology, in particular to polypeptide, recombinant XIX type humanized collagen and application thereof. Background Collagen is a protein widely distributed in connective tissues of human bodies, is also protein with the largest content of human bodies, and can account for 25% -35% of the total protein. At least 28 collagen subtypes are found in human body, and are respectively located in different tissues and organs. However, the natural collagen is low in the living body, the extraction process is complex, and animal-derived immune response is also an important cause of limited application of the collagen. With the development of genetic engineering techniques, one can construct recombinant collagen by selecting a short amino acid sequence from natural human collagen. The recombinant collagen has the advantages of low immunogenicity, high bioactivity, high stability and the like. With the increasing growth of the collagen industry, collagen obtained by using a biosynthesis pathway is becoming mature, especially humanized collagen. Recombinant humanized collagen refers to the full-length or partial amino acid sequence functional region encoded by a specific type of human collagen gene prepared by DNA recombination techniques, or a combination of functional regions containing human collagen functions. The XIX type collagen is a collagen existing in human bodies, is relatively less studied, but has been found to have the following effects and functions that 1. The XIX type collagen can maintain the structure and functions of tissue organs, namely, the XIX type collagen is one of the components of a basement membrane, plays an important role in the tissue organs such as skin, eyes, kidneys and the like, helps to maintain the integrity and stability of the basement membrane, and provides support and fixation for cells, thereby ensuring the normal structure and functions of the tissue organs. For example, in skin, type XIX collagen, along with other collagens, participates in the scaffold structure that makes up the skin, helping to maintain the elasticity and toughness of the skin. 2. Involved in cell signaling, XIX-type collagen can interact with receptors on the cell surface, and participate in the cell signaling process. The XIX type collagen can regulate the proliferation, differentiation, migration, survival and other behaviors of cells, and has important significance on the development, repair and regeneration of tissues. Such as influencing the behavior of cells during the wound healing process, promoting proliferation and migration of cells around the wound, and accelerating wound healing. 3. The research shows that the XIX type collagen plays a certain role in the angiogenesis process. Can regulate vascular endothelial cell behavior, promote vascular endothelial cell proliferation and migration, and thus facilitate formation of new blood vessel. The method is of great importance to the nutrition supply of tissues and the discharge of metabolic waste, and has great significance in the processes of embryo development, tissue repair, tumor growth and the like. While type XIX collagen has these potential effects and roles, its research is still in progress and many specific mechanisms and functions are not yet fully defined. In the prior art, the research on the recombinant XIX type collagen is less, and the recombinant collagen has the problems of poor stability and the like caused by unmodified and non-formed advanced triple helix conformation after translation, low bioactivity and no helix structure, so that the application of the recombinant collagen in the fields of cell adhesion, proliferation and the like is limited. Therefore, it is necessary to study novel XIX-type collagen. Disclosure of Invention It is an object of the present invention to provide a protein having a good cell adhesion activity, no cytotoxicity, and a typical triple helix structure. The invention provides a polypeptide, the amino acid sequence of which comprises n repeating units; The repeating unit comprises any one of the following amino acid sequences (1) - (3) or variant sequences thereof: (1) An amino acid sequence as set forth in any one of SEQ ID NO. 1-2; (2) An amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID nos. 1-2; (3) A variant sequence obtained by mutating one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NO. 1-2; wherein n is more than or equal to 1 and is an integer. The invention develops polypeptide which has human XIX type collagen triple helix structure and can perform the function of human collagen by applying the technology of synthetic biology and structural biology, has good cell adhesion activity and no cytoto