Search

CN-121974985-A - Polypeptide, recombinant X-type humanized collagen and application thereof

CN121974985ACN 121974985 ACN121974985 ACN 121974985ACN-121974985-A

Abstract

The invention relates to the technical field of biology, and particularly discloses a polypeptide, recombinant X-type humanized collagen and application thereof. The invention provides a polypeptide which can effectively promote cell adhesion and promote cell proliferation activity. Specifically, the amino acid sequence of the polypeptide comprises n repeating units, the amino acid sequence of the repeating units comprises any one of (a) a sequence shown as SEQ ID NO.1 or SEQ ID NO.2, (b) a sequence which is obtained by adding, substituting and/or deleting 1 or more amino acid residues in the sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and retains a biological activity function, and (c) a sequence which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO.1 and retains a biological activity function. The invention provides a new method for cartilage repair.

Inventors

  • LAN XIAOBIN
  • LI YUAN
  • ZHOU WENRONG
  • WANG LINGLING
  • ZHANG YONGJIAN
  • Yin Lilan
  • WANG YAJUN
  • LIU XIN
  • ZHANG XIAOBO
  • YANG XIA
  • YANG JINGBO
  • Deng Pengjun
  • LIU ZENGYAO
  • SONG HAIHONG
  • WANG YING

Assignees

  • 山西锦波生物医药股份有限公司

Dates

Publication Date
20260505
Application Date
20251210

Claims (19)

  1. 1. A polypeptide, characterized in that the amino acid sequence of the polypeptide comprises n repeated units, n is an integer greater than or equal to 1, when n is an integer greater than or equal to 2, the repeated units are directly connected or connected through connecting peptide, wherein the amino acid sequence of the repeated units comprises any one of the following (a) - (c): (a) A sequence shown as SEQ ID NO.1 or SEQ ID NO. 2; (b) A sequence which adds, substitutes and/or deletes 1 or more amino acid residues in the sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and retains the function of biological activity; (c) A sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence as set forth in SEQ ID No.1 and retaining biologically active function.
  2. 2. The polypeptide according to claim 1, wherein the amino acid sequence of the repeating unit comprises a sequence which adds, replaces and/or deletes 12-66 amino acid residues in the sequence shown as SEQ ID No.2 and retains biologically active functions; preferably, the amino acid sequence of the biologically active functional sequence has at least 80%, 85%, 90%, 95%, 98% or 100% identity to the sequence shown in SEQ ID NO. 16.
  3. 3. The polypeptide according to claim 2, characterized in that the amino acid sequence of the repeating unit is extended by an integer of 1 to 54 amino acids at the N-and/or C-terminus based on SEQ ID NO.16, preferably by 1, 12, 48 or 54 amino acids.
  4. 4. A polypeptide according to claim 3, wherein the amino acid sequence of the repeat unit is selected from any one of the group: (1) A sequence of 54 amino acids extended at the N-terminus and 12 amino acids extended at the C-terminus on the basis of SEQ ID No.16 as shown in SEQ ID No.3, or a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retaining biologically active function; (2) A sequence of 48 amino acids extended at the N-terminus and 12 amino acids extended at the C-terminus on the basis of SEQ ID No.16 as shown in SEQ ID No.4, or a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retaining biologically active function; (3) A sequence of 12 amino acids extended C-terminally on the basis of SEQ ID No.16 as shown in SEQ ID No.5, or a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retaining biologically active functions.
  5. 5. The polypeptide according to any one of claims 1 to 4, characterized in that n is an integer between 1 and 20, preferably an integer between 4 and 12, more preferably 4 or 12; preferably, when n≥2, the repeating units are directly linked.
  6. 6. The polypeptide according to any one of claims 1 to 5, characterized in that it has an amino acid sequence as shown in any one of SEQ ID nos. 6 to 10.
  7. 7. A fusion protein, characterized in that the fusion protein comprises the polypeptide of any one of claims 1-6, preferably obtained by fusing the polypeptide with other functional polypeptides.
  8. 8. A recombinant X-type humanized collagen, characterized in that the recombinant X-type humanized collagen comprises the polypeptide of any one of claims 1-6; Preferably, the recombinant X-type humanized collagen has cell adhesion and/or cell proliferation promoting activity, more preferably, has biological activity on chondrocytes; Preferably, the recombinant X-type humanized collagen has a trimeric and/or triple helix structure.
  9. 9. A polynucleotide encoding the polypeptide of any one of claims 1 to 6 or the fusion protein of claim 7 or the recombinant humanized X collagen of claim 8; Preferably, the polynucleotide comprises the nucleotide sequence set forth in any one of SEQ ID NOS.11-15 or a degenerate sequence thereof.
  10. 10. The polynucleotide according to claim 9, wherein the polynucleotide further comprises a sequence encoding a purification tag and/or a leader sequence, preferably the purification tag is selected from the group consisting of His tag, GST tag, MBP tag, SUMO tag or NusA tag.
  11. 11. A biological material, characterized in that it comprises the polynucleotide of claim 9 or 10; the biological material is an expression cassette, a vector or a host cell; Preferably, the vector is an expression vector, preferably further comprising a control element operably linked to the polynucleotide, preferably a promoter, terminator and/or enhancer; Preferably, the host cell is a bacterium, a fungus or an animal cell, wherein the bacterium preferably comprises E.coli, and the fungus preferably comprises yeast, more preferably Saccharomyces cerevisiae or Pichia pastoris.
  12. 12. The method for preparing the polypeptide according to any one of claims 1 to 6 or the fusion protein according to claim 7 or the recombinant X-type humanized collagen according to claim 8, wherein the method comprises culturing host cells comprising the polynucleotide according to claim 9 or 10, harvesting host cells and/or culture solution comprising the polypeptide or the fusion protein or the recombinant X-type humanized collagen, and separating the polypeptide or the fusion protein or the recombinant X-type humanized collagen from the host cells and/or culture solution.
  13. 13. A composition comprising one or more polypeptides according to any one of claims 1 to 6, fusion proteins according to claim 7, recombinant humanized X collagen according to claim 8, polynucleotides according to claim 9 or 10, and biological material according to claim 11.
  14. 14. The composition of claim 13, wherein the composition is selected from the group consisting of biological dressing, biomimetic material of the human body, cosmetic material of plastic, organoid culture material, cardiovascular scaffold material, coating material, tissue filling/compatibilizing material, ophthalmic material, obstetrical and gynecological biological material, nerve repair regenerating material, chronic wound repair material, bone or cartilage regenerating material, liver tissue material, vascular repair regenerating material, 3D printed artificial organ biological material, cosmetic raw material, pharmaceutical adjuvant, pharmaceutical or food additive; preferably, the composition is a topical composition, an injectable composition or an oral composition; Preferably, the composition is in the form of a solution, lyophilized powder, gel, sponge or fiber.
  15. 15. Use of the polypeptide of any one of claims 1-6, the fusion protein of claim 7, the recombinant X-type humanized collagen of claim 8, the polynucleotide of claim 9 or 10, the biological material of claim 11 or the composition of claim 13 or 14 for the preparation of a biological dressing, a human biomimetic material, a cosmetic material for plastic, an organoid culture material, a cardiovascular scaffold material, a coating material, a tissue filling/compatibilizing material, an ophthalmic material, a gynaecological biological material, a nerve repair regeneration material, a chronic wound repair material, a muscle or bone or cartilage or ligament regeneration material, a liver tissue material, a vascular repair regeneration material, a 3D printing artificial organ biological material, a cosmetic material, a pharmaceutical adjuvant, a pharmaceutical or a food additive.
  16. 16. Use of the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant humanized collagen type X of claim 8, the polynucleotide of claim 9 or 10, the biomaterial of claim 11 or the composition of claim 13 or 14 for the preparation of a product for any one or more of cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or compatibilization, preferably, tissue repair is cartilage repair.
  17. 17. Use of the polypeptide according to any one of claims 1 to 6, the fusion protein according to claim 7 or the recombinant X-type humanized collagen according to claim 8 for any one or more of the following purposes selected from cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or compatibilization, cosmetic shaping, and for non-disease diagnosis or treatment.
  18. 18. Use of the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant humanized collagen type X of claim 8, the polynucleotide of claim 9 or 10, the biomaterial of claim 11 or the composition of claim 13 or 14 for the preparation of a medicament for preventing and/or treating a disease or disorder associated with defects in collagen type X or for supplementing collagen type X.
  19. 19. A method for promoting cell adhesion and/or proliferation and/or improving extracellular matrix, characterized in that it comprises the step of contacting a polypeptide according to any one of claims 1 to 6, a fusion protein according to claim 7, a recombinant humanized collagen type X according to claim 8 or a composition according to claim 13 or 14 with a cell, preferably an animal cell, more preferably a mammalian cell, still more preferably a human cell, for non-disease diagnostic or therapeutic purposes; preferably, the promotion of cell adhesion and/or proliferation and/or improvement of extracellular matrix is performed in vivo/in vitro, wherein the improvement of extracellular matrix refers to promotion of extracellular matrix to form a network structure.

Description

Polypeptide, recombinant X-type humanized collagen and application thereof Technical Field The invention relates to the technical field of biology, and relates to a polypeptide, recombinant X-type humanized collagen and application thereof, in particular to the polypeptide, the recombinant X-type humanized collagen and application thereof in cartilage repair. Background Collagen is protein with the largest content in human body, accounting for 25% -35% of the total protein, and is widely distributed in connective tissues, at least 28 subtypes exist, and the collagen is respectively distributed in different tissues. Type X collagen (Type X collagen) is a short chain non-fibrillar collagen, whose molecular structure consists of three identical α1 (X) chains forming a homotrimer, with a triple helical domain length of 132 nm, which is only half of Type II collagen, but retains the larger non-collagenous globular domain at the C-terminus. The human COL10A1 gene is located on the long arm of chromosome 6 (6 q21-q 22.3), and its coding region is highly compact and is mainly distributed over two exons, one 169 bp encoding the signal peptide and the N-terminal non-collagenous domain, and the other approximately 2940 bp encoding the remaining N-terminal domain, the complete triple helix domain (463 amino acids), the C-terminal domain (161 amino acids) and the 3' untranslated region. The expression of type X collagen has strict space-time specificity and is produced only by hypertrophic chondrocytes in endochondral ossified regions (e.g., the hypertrophic zone of growth plates). Immunolocalization studies showed that it exhibited a narrow band-like distribution in the growth plates of human fetal long bones and ribs, closely overlapping the calcification fronts. Of particular note, collagen type X was synthesized earlier than mineral deposition, suggesting that it may be involved in initiating the mineralization process. Specifically, in fetal cartilage, the immunoreactivity of type X collagen precedes the appearance of calcium signal detected by alizarin red S, which discovery provides direct evidence for its precursor role in mineralization. The unique properties of type X collagen offer a number of possibilities for its use in the medical field. In the case of bone diseases, its specific expression pattern in growth plates can be used as a molecular marker of the endochondral ossification process. In the intervertebral disc of osteoarthritis patients, changes in the expression of type X collagen may serve as biomarkers of disease progression or repair. Based on the gene regulation mechanism, the cis-regulatory element of the COL10A1 gene can be used as an intervention target. In the field of regenerative medicine, the extraction and purification technology of the X-type collagen lays a foundation for developing cartilage repair materials, and the controllable degradation characteristic (such as 53 kDa reduced by pepsin treatment) can be used for designing biological materials with specific release performance. Therefore, there is a need to explore further effective ways of exogenously supplementing or filling type X collagen. In the prior art, there are methods for preparing the type X collagen peptide by using animal-derived biological materials (such as eggshell membrane extract) as raw materials, but the method has limited use because the source of the collagen peptide is non-human and can cause immune safety problems when the collagen peptide is practically applied to bone tissues. Therefore, developing an X-type collagen product suitable for mass production and having practical application potential without immunogenicity risk has become a research direction to be advanced in the field. Disclosure of Invention The invention aims to provide recombinant X-type humanized collagen which has high safety and can effectively promote cell proliferation and adhesion. The invention provides a polypeptide, the amino acid sequence of which comprises n repeated units, n is an integer greater than or equal to 1, when n is an integer greater than or equal to 2, the repeated units are directly connected or are connected through connecting peptide (one or more amino acid residues are separated between the repeated units), wherein the amino acid sequence of the repeated units comprises any one of the following (a) - (c): (a) A sequence shown as SEQ ID NO.1 or SEQ ID NO. 2; (b) A sequence which adds, substitutes and/or deletes 1 or more amino acid residues in the sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and retains the function of biological activity; (c) A sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence as set forth in SEQ ID No.1 and retaining biologically active function. In the polypeptide of the invention, the amino acid sequence of the repeating unit comprises a sequence in which 12-66 amino acid residues (in certain embodiments, the number of ami