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CN-120623318-B - Recombinant human XVII type collagen fragment based on natural collagen sequence, and preparation method and application thereof

CN120623318BCN 120623318 BCN120623318 BCN 120623318BCN-120623318-B

Abstract

The application provides a recombinant XVII type collagen fragment, the protein fragment sequence of which is shown as SEQ ID No.1, and the recombinant XVII type collagen fragment is designed based on natural collagen sequence screening, has a triple helix structure, has good biological activity, can effectively play a role, and has good promotion effect on cell migration, cell proliferation and cell adhesion. The method for preparing the recombinant XVII type collagen fragment has simplified process steps, improves the expression efficiency of recombinant protein and reduces the production cost. The recombinant XVII type collagen fragment has good mechanical property and biological activity, and can be used for preparing therapeutic drugs and biological materials.

Inventors

  • ZHANG DELIN
  • WANG XIANGYI
  • NIU SHUHUI
  • SUN FANG
  • YIN PING
  • LI GUOLIN

Assignees

  • 深圳市成美生物科技有限公司

Dates

Publication Date
20260512
Application Date
20250612

Claims (19)

  1. 1. The recombinant XVII type collagen fragment has a protein fragment sequence shown in SEQ ID No. 1.
  2. 2. A nucleic acid encoding the recombinant XVII collagen fragment according to claim 1.
  3. 3. An expression vector having the nucleic acid of claim 2 recombined thereon.
  4. 4. The vector of claim 3, wherein the expression vector comprises pCM01-COL17-1.
  5. 5. An expression cell comprising the nucleic acid of claim 2 or comprising the expression vector of claim 3.
  6. 6. The expression cell of claim 5, wherein the expression cell comprises a pichia strain.
  7. 7. A method of producing a recombinant XVII type collagen fragment, comprising inoculating the expression cell of claim 5 into a culture medium to express said recombinant XVII type collagen fragment.
  8. 8. The method of claim 7, wherein the medium comprises 1-9g/L potassium dihydrogen phosphate, 0.1-5g/L calcium sulfate dihydrate, 5-15g/L magnesium sulfate heptahydrate, 5-15g/L potassium sulfate, 10-80g/L ammonium dihydrogen phosphate, 0.1-5g/L potassium hydroxide, 5-25g/L glycerol.
  9. 9. The method of claim 7, wherein the medium comprises 4-5g/L potassium dihydrogen phosphate, 0.5-1g/L calcium sulfate dihydrate, 8-10g/L magnesium sulfate heptahydrate, 8-10g/L potassium sulfate, 30-40g/L ammonium dihydrogen phosphate, 1-2g/L potassium hydroxide, and 10-15g/L glycerol.
  10. 10. The method of claim 7, wherein the medium comprises 5g/L potassium dihydrogen phosphate, 1g/L calcium sulfate dihydrate, 10g/L magnesium sulfate heptahydrate, 10g/L potassium sulfate, 40g/L ammonium dihydrogen phosphate, 2g/L potassium hydroxide, 15g/L glycerol.
  11. 11. The method of claim 7, wherein glycerol feed is used during the strain growth phase when the expression cells are inoculated into the culture medium.
  12. 12. The method of claim 7, wherein the recombinant XVII type collagen fragment is induced to be expressed using methanol after the end of the growth of the expression cells.
  13. 13. The method of claim 7, wherein the expression cells are inoculated at 10% v/v; Or the culture medium also contains 24mg/L CuSO 4 ·5H 2 O、12 mg/L MnSO4·H2O、260mg/L FeSO 4 ·7H 2 O、80 mg/L ZnSO 4 ·7H 2 O、0.8 mg/L CoC1 2 、0.08 mg/L H 3 BO 3 、0.8 mg/L NaMoO 4 ·2H 2 O、0.4 mg/L KI、0.8 mg/L biotin with final concentration of 20 mg/L H 2 SO 4 ; Or the fermentation condition of the growth stage of the expression cells is that the temperature is 30 ℃ and the pH is 4.5; or the fermentation condition of the recombinant XVII type collagen fragment in the induction expression stage is at the temperature of 28 ℃ and the pH value of the recombinant XVII type collagen fragment is 5.0; Or when the wet weight of the cell body of the expression cell reaches 180g/L-220g/L, starting to induce methanol.
  14. 14. The method of claim 7, further comprising a purification step of separating and purifying the supernatant after the fermentation reaction.
  15. 15. The method of claim 14, wherein the purifying step comprises purifying using weak cation exchange chromatography followed by further purification using hydrophobic chromatography to obtain the recombinant XVII type collagen fragment.
  16. 16. The method of claim 14, wherein the purification step is followed by a concentration step.
  17. 17. A composition or article comprising the recombinant XVII collagen fragment according to claim 1.
  18. 18. The composition or article of claim 17, wherein the composition is a skin care or pharmaceutical composition and the article is one or more of a medical device, a biological material, a tissue engineering product, a cosmetic, a health care product.
  19. 19. Use of the recombinant XVII collagen fragment according to claim 1 or the recombinant XVII collagen fragment prepared by the method according to any one of claims 7 to 16 for the preparation of a medicament or product for skin basement membrane repair, epidermis anti-aging, skin wound repair.

Description

Recombinant human XVII type collagen fragment based on natural collagen sequence, and preparation method and application thereof Technical Field The invention relates to the field of biotechnology, in particular to a method for screening, optimizing and preparing a recombinant human XVII type collagen fragment based on a natural collagen sequence, the obtained recombinant human XVII type collagen fragment and application of the protein fragment in the field of biomedicine. Background The XVII type collagen is a transmembrane protein, which is mainly distributed in the junction region between epithelial cells and dermal cells, and is involved in maintaining the integrity of extracellular matrix and cell signaling. In dermatology, wound healing and inflammation related diseases, collagen type XVII has an important role. However, current research on this protein is focused mainly on functional analysis of full-length proteins, and lack of screening and optimization for functional fragments thereof. Therefore, the recombinant human XVII type collagen with enhanced biological activity prepared by screening the natural collagen sequence is developed, and has important scientific significance and application value. Bovine type I collagen is the most widely used natural collagen material at present, and is widely used in tissue repair, wound dressing, injection filling, cell culture scaffold and skin care products due to its stable source, low cost and structure close to human collagen. Several researches show that the bovine type I collagen can promote cell adhesion, proliferation and tissue regeneration to a certain extent, particularly has good performance in promoting wound closure and cell adhesion, and is one of collagen materials with higher maturity in the current clinical and cosmetic application due to the good extracellular matrix structural support. However, bovine collagen has relatively weak pro-cell migration and proliferative activity, which is a limiting factor in situations where rapid cellular response and tissue regeneration are required (e.g., chronic wounds, dermis layer repair). Meanwhile, bovine-derived collagen belongs to heterologous protein, has a certain immunogenicity risk and potential animal-derived pollution problem, and has use limitation in the development of novel medical or cosmetic products with higher safety requirements. In recent years, recombinant human XVII type collagen has gradually entered the development stage, and according to the published patent data (such as CN116640205A, CN119462893A, CN 119504982A), various XVII type fragment molecules have been selected for tissue repair, wound dressing, injection filling, cell culture scaffolds and skin care products. The CJ-XVII type XVII collagen (hereinafter referred to as Biddish product 1) developed by Innovative medical science and technology Co., ltd is the main stream of XVII type collagen molecules (disclosed in CN 113185604B) in the current market, and various studies and product data show that Biddish product 1 has good cell adhesion and cell migration promoting capabilities, but the cell proliferation promoting effect is not reported, and the stability and structure of the collagen have not been confirmed although the collagen has amide I, II and III characteristic infrared spectra. The XVII fragment which has high bioactivity, high thermal stability and complete triple helix structure has not been developed yet, and cannot provide a basis for further improving the application of collagen. Based on the current technical development in the art, there is a need to provide human XVII type collagen with high thermal stability, complete triple helix structure and good bioactivity. Disclosure of Invention The application aims to provide a preparation method of a recombinant human XVII type collagen fragment screened and optimized based on a natural collagen sequence, so as to obtain a functional protein fragment with high activity, high stability and easy expression. Furthermore, the application relates to potential applications of the protein in biomedical fields, including but not limited to therapeutic proteins, diagnostic reagents and drug screening platforms. Based on this, a first aspect of the present application is to provide a recombinant XVII-type collagen fragment, the protein fragment sequence of which is shown in SEQ ID No. 1. The XVII type Collagen (Collagen XVII, also called COL17/BP180/BPAG 2) is a transmembrane protein with a unique key structure and located between epidermis and dermis, has important effects on basement membrane repair and epidermis anti-aging, is a key factor for skin aging and wound repair, maintains skin in a 'young state', maintains hair follicle stem cells (alopecia, poliosis and the like), and plays an important role in the skin wound repair process by affecting migration, proliferation and differentiation of the stem cells. The recombinant XVII type collagen fragment of the applica