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CN-121975002-A - Recombinant fully humanized XVII type collagen, expression system and application thereof

CN121975002ACN 121975002 ACN121975002 ACN 121975002ACN-121975002-A

Abstract

The invention discloses a recombinant fully humanized XVII type collagen, an expression system and application thereof. Wherein the recombinant fully humanized XVII type collagen is recombinant protein C17-1, C17-2 or C17-3, the amino acid sequence of the recombinant protein C17-1 is shown as SEQ ID NO.1, the amino acid sequence of the recombinant protein C17-2 is shown as SEQ ID NO.3, and the amino acid sequence of the recombinant protein C17-3 is shown as SEQ ID NO. 5. The invention can adopt an escherichia coli expression system, and can perform high-density fermentation by adding the fusion expression of the soluble tag protein and the preferable XVII type collagen multi-fragment, so that the high-solubility XVII type collagen can be expressed in a large scale, low cost and high efficiency way. The recombinant XVII type collagen and the fully humanized expression system thereof can be used as key raw materials in the fields of cosmetics or medical appliances.

Inventors

  • FU HAILONG
  • WANG ZHILIN

Assignees

  • 常州耀宏生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260115

Claims (13)

  1. 1. A recombinant fully humanized XVII type collagen is characterized in that the recombinant XVII type collagen is recombinant protein C17-1, C17-2 or C17-3, the amino acid sequence of the recombinant protein C17-1 is shown as SEQ ID NO.1, the amino acid sequence of the recombinant protein C17-2 is shown as SEQ ID NO.3, and the amino acid sequence of the recombinant protein C17-3 is shown as SEQ ID NO. 5.
  2. 2. The recombinant fully humanized XVII collagen according to claim 1, wherein the DNA sequence of recombinant protein C17-1 is shown in SEQ ID NO.2, the DNA sequence of recombinant protein C17-2 is shown in SEQ ID NO.4, and the DNA sequence of recombinant protein C17-3 is shown in SEQ ID NO. 5.
  3. 3. A recombinant collagen expression vector, characterized in that the vector contains the DNA sequence of the recombinant protein C17-1, C17-2 or C17-3 of claim 1.
  4. 4. A recombinant collagen expression vector according to claim 3, characterized in that the vector is a plasmid, in particular a pET22B plasmid.
  5. 5. A recombinant collagen having a lytic fusion tag, comprising a lytic fusion tag portion and a recombinant fully humanized XVII collagen portion according to claim 1, wherein said lytic fusion tag is selected from the group consisting of histidine tag His, thioredoxin Trx, small molecule ubiquitin-related modifier protein Sumo, maltose binding protein MBP, glutathione S transferase GST, disulfide bond forming protein DsbA, disulfide bond forming protein DsbC, and N-utilizing substance a NusA protein.
  6. 6. Recombinant collagen having a lytic fusion tag according to claim 5, characterized in that the lytic fusion tag is selected from Sumo protein tags.
  7. 7. The recombinant collagen with the fusion tag of claim 6, wherein the recombinant collagen with the fusion tag of the protein is protein SC17-1, SC17-2 or SC17-3 respectively, wherein the amino acid sequence of the protein SC17-1 is shown as SEQ ID NO.10, the amino acid sequence of the protein SC17-2 is shown as SEQ ID NO.12, and the amino acid sequence of the protein SC17-3 is shown as SEQ ID NO. 14.
  8. 8. The recombinant collagen with a fusion tag of a pro-lytic protein according to claim 7, wherein the DNA sequence of the protein SC17-1 is shown in SEQ ID NO.11, the DNA sequence of the protein SC17-2 is shown in SEQ ID NO.13, and the DNA sequence of the protein SC17-3 is shown in SEQ ID NO. 15.
  9. 9. An escherichia coli capable of expressing XVII collagen, which is characterized in that the escherichia coli contains the DNA sequence of the recombinant fully humanized XVII type collagen as set forth in claim 1 or is obtained by transforming an escherichia coli strain, in particular an escherichia coli Bl21 (DE 3) strain, with the recombinant collagen expression vector as set forth in claim 3.
  10. 10. A P4HA1 gene based on an escherichia coli co-expression system and used for carrying out hydroxyproline modification on XVII type collagen is characterized in that the DNA sequence of the P4HA1 gene is shown as SEQ ID NO. 16.
  11. 11. An escherichia coli capable of directly expressing hydroxyproline modified XVII collagen, which is characterized by comprising the DNA sequence of the recombinant fully humanized XVII collagen according to claim 1 and the DNA sequence of the P4HA1 gene according to claim 10.
  12. 12. Use of recombinant fully humanized XVII collagen or recombinant fully humanized XVII collagen modified by hydroxyproline in the preparation of a product for promoting wound healing, in the preparation of a product for promoting soft tissue regeneration, in the preparation of a product for promoting hair growth, in the preparation of a product for promoting cell repair, as a cosmetic additive or as a functional food additive, wherein the recombinant fully humanized XVII collagen is the recombinant protein C17-1, C17-2 or C17-3 according to claim 1.
  13. 13. A method for purifying recombinant fully humanized XVII type collagen according to claim 1 is characterized in that recombinant collagen with an osteolytic fusion tag according to claim 5 is taken, or a solution prepared by fermentation of recombinant bacteria and containing the recombinant collagen with the osteolytic fusion tag according to claim 5 is taken, and is mixed with ulp protease, then enzyme digestion is carried out, and the product after enzyme digestion is purified and separated, so that the recombinant fully humanized XVII type collagen is obtained, wherein the mass and the dosage ratio of ulp protease to the recombinant collagen with the osteolytic fusion tag is 1:1000-1:10000, the enzyme digestion temperature is 1-10 ℃, and the enzyme digestion time is 6-12 hours.

Description

Recombinant fully humanized XVII type collagen, expression system and application thereof Technical Field The invention belongs to the field of recombinant protein materials, and particularly relates to recombinant fully humanized XVII type collagen, an expression system and application thereof, in particular to recombinant collagen, an expression system thereof, a preparation method and application thereof. Background Collagen is the most important protein in animal connective tissue, and is the most abundant protein in human body, and accounts for 25% -30% of the total protein. As an important macromolecular biological protein, collagen and its degradation products (polypeptides) have proven to be powerful chemokines in the medical field, particularly in promoting wound healing and soft tissue regeneration. As an active biological material, collagen has been used for many years in the fields of various medical devices such as wound dressing, skin repair and medical injection. In addition, the collagen can be used in a plurality of fields such as cosmetic additives (skin care products, facial masks and the like), functional foods and the like, and has wide application prospect. The industrially prepared collagen mainly has two sources, namely, the collagen is prepared by natural extraction from animal tissues and recombination by utilizing a genetic engineering technology. The extraction of collagen from animal tissues is still the main production method of medical collagen materials, the extraction objects comprise cows, pigs, fish and the like, and mammals, especially cows, are still the main material sources, always occupy more than 1/3 market share, and the ratio is improved year by year. The animal collagen extraction method has two methods, namely an acid method and an enzyme method, and is characterized in that the triple helix structure is clear and complete, and the animal collagen extraction method has better biological activity. However, animal collagen extraction has long extraction period and low yield, may have the risk of acid solvent residue, and a large number of non-collagen peptide fragments may be retained in the treatment process, so that the animal collagen extraction has potential safety problems such as immunogenicity and virus epidemic disease. Meanwhile, the production of recombinant human collagen by a genetic engineering method is gradually rising in recent years and has realized industrial production, and the genetic engineering method has also been developed in the aspects of verification of the structure and activity of the recombinant collagen. Type XVII collagen (Collagen Type XVII) is a transmembrane collagen, encoded by the COL17A1 gene for 1497 amino acids, the N-terminus of which is located in the cytoplasm and the C-terminus of which is located in the extracellular matrix, joined by a short intermediate transmembrane sequence. Type XVII collagen is expressed primarily in skin, hair follicles, and other tissues associated with the epithelial-mesenchymal interface. The XVII type collagen is mainly present in the basal membrane strip and is responsible for tightly connecting the epidermis and dermis together, which is an important factor for maintaining the overall structural stability of the skin. Human XVII collagen can effectively regulate the adhesion, proliferation and migration of fibroblasts, and promote the differentiation and regeneration of keratinocytes. In addition, studies have shown that XVII type collagen also plays an important role in hair growth, having a variety of functions including maintaining the hair follicle stem cell microenvironment, regulating the hair follicle cycle, promoting hair follicle development and regeneration, and maintaining the skin barrier. The systems that can be used for recombinant production of collagen include mammalian cells, insect animal cells, yeast and E.coli expression systems. Because of the transmembrane nature of XVII collagen and its non-fibroblastic collagen structure, recombinant preparation, especially of the full length or entire extracellular domain, is very difficult on a large scale. Early recombinant preparation mainly uses human cells or mammalian cell expression systems such as CHO to prepare micro proteins for related disease pemphigus research and collagen basement membrane binding research. The mammalian cell system in the industry is mainly used for expressing medicines with higher value, and the culture medium has high cost and is not suitable for preparing collagen. The insect animal cell expression system is mostly used for preparing a small amount of protein in the scientific research field, and is not suitable for preparing large-scale biological products. Thus, expression systems for recombinant collagen are mainly yeast systems and E.coli systems. The yeast system is used as a eukaryotic expression system, has certain post-translational processing capacity, and the prepared protein has certain p