Biosynthetic manufacturing method for human body structural materials

Abstract

This specification provides a method for the biosynthesis and production of human body structural materials. The polypeptide has the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6. The recombinant humanized type VII collagen produced herein has high cell proliferation-promoting activity and does not induce an immune response when administered to the human body. Furthermore, the novel production method allows for large-scale production of recombinant humanized type VII collagen, making it widely applicable to the production of human body structural materials. Applications include the production of high-end medical devices, such as bio-dressings, human biomimetic materials, cosmetic and cosmetic materials, organoid culture materials, tissue injection and filling materials, skin repair and regeneration materials, oral mucosal repair and regeneration materials, cervical mucosal repair and regeneration materials, gynecological biomaterials, and 3D-printed artificial organ biomaterials, as well as the production of high-end cosmetic raw materials, high-end pharmaceutical supplements, and food additives.

Description

[Technical Field]

[0001] cross reference This application claims priority from a Chinese patent application filed on August 23, 2022, bearing application number 202211017546.9 and entitled "Bio-synthetic manufacturing method for human body structural materials," which is incorporated herein by reference.

[0002] Technical Field This application belongs to the technical field of synthetic biology and relates to a method for the biosynthetic production of structural materials for the human body. [Background technology]

[0003] Human structural materials contain structural proteins, primarily collagen, which have adhesive and supportive functions for cells and tissues and are the primary components of the extracellular matrix.

[0004] Collagen is a protein widely distributed in the connective tissues of the human body. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein content. Currently, at least 28 collagen subtypes have been found in the human body, each located in different tissues and organs. Type VII collagen is a fibrillar collagen found in the basement membrane region of stratified squamous epithelia, such as the skin, oral mucosa, and cervix, and is therefore also known as basement membrane collagen. Type VII collagen is the main component of anchoring fibrils in the skin. These fibrils extend from the lamellar dendrites of the epidermal basement membrane to the dermal connective tissue, providing adhesion between the epidermis and dermis. Animal experiments have demonstrated that type VII collagen, through tissue laminin, promotes fibrocyte migration and cytokine secretion, contributing to skin damage and repair. Artificial injection of recombinant type VII collagen can correct dystrophic epidermal necrosis (bullous epidermal necrosis).

[0005] Currently, crude type VII collagen is primarily obtained through extraction from animal tissues or delayed viral transfection. Unfortunately, type VII collagen has a low biological content, the extraction process is complicated, and the animal-derived immune response is a major factor limiting its application. While delayed viral transfection has relatively low immunogenicity, it is difficult to operate and has significant limitations on target gene capacity. With the continued expansion of China's collagen industry, biosynthetic collagen production has become increasingly mature, with humanized collagen in particular leading the way globally. In 2021, the National Medical Products Administration of China established a nomenclature classification for biosynthetic collagen. Recombinant humanized collagen refers to a full-length or partial amino acid sequence encoded by a specific human collagen gene, or a combination of fragments containing human collagen functions, produced through DNA recombinant technology.

[0006] Type VII collagen is a homotrimer consisting of three identical α1 chains, with the molecular structure α1α1α1(VII). Three α1(VII) chains twist together to form three strand-like molecules of procollagen. Procollagen molecules are secreted from cells and undergo enzymatic treatment to remove excess protein fragments from their ends. After processing, procollagen molecules arrange themselves into thin, undeveloped bundles to form mature type VII collagen. Each α1 polypeptide chain contains a central collagenous triple-helical region flanked by non-collagenous amino and carboxy termini. The collagenous structural region is a triple-helical domain consisting of a characteristic Gly-XY repeat sequence. Genetic mutations in type VII collagen cause impaired collagen synthesis or structural abnormalities, leading to various levels of blistering, which can manifest in sites such as the skin, oral mucosa, and esophagus. While most mutations are nonsense, missense mutations still occur, causing collagen retention and misfolding within cells, affecting the connection between the vaginal mucosa and the lamina propria of the basement membrane.

[0007] Currently, type VII collagen is mainly obtained by enzymatic hydrolysis and delayed viral transfection. Enzymatic hydrolysis involves treating animal tissues with proteases to extract and obtain type VII collagen derivatives. However, the collagen extracted by this method has already lost its original biological activity and is unable to exert its true function. Delayed viral transfection involves constructing a retroviral vector, transfecting cells, and finally purifying the resulting type VII collagen. However, this manufacturing process suffers from several drawbacks: high production difficulty, difficulty in obtaining highly purified viruses, random integration of the delayed viral vector into the host genome, which may interfere with the expression of genes at and near the insertion site, difficulty in precisely controlling the integrated copy number, and small target gene capacity. Therefore, a biosynthetic method for recombinant humanized type VII collagen that overcomes these drawbacks is needed to enable its widespread application as a human body structural material. Summary of the Invention

[0008] In response to the deficiencies of the prior art, the present application has designed a screening and synthesis process for the core functional domain of recombinant humanized collagen type VII. The present application is the first invention to design a screening and protein synthesis process for the functional domain of recombinant humanized collagen type VII.

[0009] In one aspect, the present application provides a repeating unit n wherein the repeating units comprise the amino acid sequence of SEQ ID NO. 1, and wherein each repeating unit is directly linked, and the number of repeating units, n, is between 4 and 20. In one embodiment, the polypeptide is recombinant humanized collagen VII.

[0010] In one embodiment, the polypeptide has the amino acid sequence of SEQ ID NO.4.

[0011] In one aspect, the present application provides a nucleic acid comprising the nucleotide sequence of a polypeptide described herein. In one embodiment, the nucleic acid further comprises a nucleotide sequence encoding a purification tag. The purification tag may be a His tag, a GST tag, a MBP tag, a SUMO tag, or a NusA tag. In one embodiment, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[0012] In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO.7.

[0013] In one aspect, the present application provides a vector comprising a nucleic acid described herein. In one embodiment, the nucleic acid can include an expression control element operably linked to the nucleic acid. In one embodiment, the expression control element can be a promoter, terminator, and / or enhancer.

[0014] In one aspect, the present application provides a host cell comprising a nucleic acid described herein or a vector described herein. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. In one embodiment, the prokaryotic cell is an E. coli cell. In one embodiment, the E. coli is E. coli BL21.

[0015] In one aspect, the present method for producing a polypeptide described herein comprises the steps of: (1) culturing a host cell described herein under suitable culture conditions; (2) harvesting the host cells and / or medium containing the polypeptide; Polypeptides and (3) purifying the above.

[0016] The purification step may be carried out by a step selected from the group consisting of (1) crude purification of the polypeptide using a Ni affinity chromatography column, (2) enzymatic cleavage by adding TEV enzyme, and (3) precision purification of the polypeptide using an ion exchange column.

[0017] In one aspect, the present application provides a composition comprising a polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a host cell described herein. For example, the composition can comprise the polypeptide. The concentration of the polypeptide can be greater than 1 mg / ml, greater than 5 mg / ml, greater than 10 mg / ml, or greater than 12 mg / ml.

[0018] In one embodiment, the composition is one or more of a bio-dressing material, a human biomimetic material, a cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a medicinal supplement, and a food additive.

[0019] In one aspect, the present application provides the use of a polypeptide as described herein, a nucleic acid as described herein, a vector as described herein and / or a host cell as described herein and / or a composition as described herein in promoting cell adhesion in vitro or in the manufacture of an article or kit for promoting cell adhesion.

[0020] In one aspect, the present application provides applications of the polypeptides described herein, the nucleic acids described herein, the vectors described herein and / or the host cells described herein and / or the compositions described herein in the manufacture of high-end medical devices, such as bio-dressing materials, human biomimetic materials, cosmetic and reconstructive materials, organoid culture materials, cardiovascular stents, coating materials, tissue injection and filling materials, ophthalmic materials, gynecological and obstetric biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, high-end cosmetic ingredients and medicinal supplement materials, and food additives.

[0021] In one aspect, the present application provides a method of promoting cell proliferation, comprising contacting a cell with a polypeptide described herein. Preferably, the cell is an animal cell, such as a mammalian cell.

[0022] Embodiments of the present application include the following.

[0023] In light of the current state of research, this application provides a method for biosynthesizing recombinant humanized type VII collagen, i.e., a method for producing a human body structural material. The specific process includes the steps of (1) screening functional regions and constructing a bacterial strain, (2) large-scale biological fermentation culture and inducible protein expression, and (3) purification and optional enzymatic cleavage of humanized type VII collagen.

[0024] According to Section 1, the screening of functional regions and the construction of strains can be carried out as follows: (1) large-scale screening of functional regions to obtain target gene fragments, (2) inserting the obtained target gene fragments into the PET-28a-Trx-His expression vector to obtain recombinant expression plasmids, and (3) introducing the recombinant expression plasmids into E. coli competent cells BL21 (DE3), and screening to obtain positive E. coli genetically engineered strains.

[0025] According to Section 1, large-scale biofermentation can be carried out as follows: The positive E. coli genetically engineered strains obtained by screening were added to the antibiotic stock shake flasks and cultured in a constant temperature shaker at 220 rpm and 37°C.

[0026] According to Section 1, induced protein expression can be performed as follows: (1) After cultivation, the shake flask was cooled to 16 to 30°C, (2) IPTG mother liquor was added to induce expression, and (3) the bacterial solution after induced expression was placed in a centrifuge flask and centrifuged at 6000 rpm at 4°C for 12 minutes, after which the bacterial cells were collected.

[0027] According to Section 1, the purification and optional enzymatic cleavage of humanized type VII collagen can be carried out as follows: (1) humanized type VII collagen is roughly purified using a Ni affinity chromatography column, (2) TEV enzyme is added at a certain ratio to perform enzymatic cleavage, and (3) humanized type VII collagen is precisely purified using an ion exchange column.

[0028] According to item 2, the functional areas screened are as follows:

[0029] Amino acid sequence of recombinant type VII humanized collagen C7P7: GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP (SEQ ID NO: 4) Amino acid sequence of recombinant type VII humanized collagen C7P11: GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPG (SEQ ID NO: 5) Amino acid sequence of recombinant type VII humanized collagen C7P12: GEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDP (SEQ ID NO: 6) .

[0030] In the present application, the codons for E. coli were optimized, and the optimized base sequence is as follows:

[0031] The nucleotide sequence of recombinant type VII humanized collagen C7P7 is GGATTTCCCGGGGTCCCGGGAGGCACCGGCCCTAAAGGCGATCGTGGTGAAACCGGCAGCAAGGGCGAGCAGGGTCTGCCGGGCGAGCGCGGTTTGAGAGGCGAACCGGGTTTTCCAGGCGTGCCGGGCGGTACGGGTCCGAAGGGTGACCGTGGCGAAACCGGCAGCAAGGGTGAACAAGGTTTACCGGGTGAACGCGGTCTGCGTGGTGAGCCGGGCTTCCCAGGTGTTCCGGGCGGAACCGGTCCTAAAGGTGATCGTGGCGAAACCGGTTCCAAAGGCGAACAAGGTCTTCCGGGTGAGCGCGGTCTGCGTGGCGAACCGGGTTTCCCGGGCGTGCCGGGAGGCACCGGCCCAAAGGGCGACCGCGGAGAAACCGGCAGCAAAGGCGAGCAGGGCCTGCCGGGTGAACGTGGCCTGCGTGGTGAGCCGGGATTCCCGGGTGTTCCGGGCGGCACCGGTCCGAAAGGTGATCGTGGTGAAACCGGTAGCAAGGGTGAACAGGGTCTGCCGGGCGAGCGCGGCTTGAGAGGTGAGCCTGGTTTTCCGGGGGTGCCCGGCGGTACGGGCCCGAAAGGCGACCGTGGCGAAACCGGTTCTAAGGGTGAGCAGGGTCTGCCGGGTGAGCGTGGTCTGCGCGGTGAGCCGGGTTTCCCGGGCGTTCCGGGTGGCACTGGTCCGAAGGGCGACCGTGGCGAGACTGGCTCGAAAGGTGAACAGGGTTTGCCGGGTGAGCGTGGTCTGCGTGGTGAGCCGGGTTTTCCGGGCGTGCCGGGTGGCACGGGCCCAAAAGGCGATCGTGGTGAGACCGGTTCCAAGGGCGAGCAAGGTCTGCCGGGCGAGCGCGGTCTCCGCGGTGAACCG (SEQ ID NO: 7) and The nucleotide sequence of recombinant type VII humanized collagen C7P11 is GGACTAACAGGGCCGACCGGTGCGGTCGGCCTGCCGGGACCACCGGGCCCCAGCGGTCTGGTTGGTCCTCAGGGTTCCCCGGGTCTTCCGGGCCAGGTTGGTGAGACAGGCAAGCCGGGTGCGCCGGGCCGTGACGGTGCCTCTGGTAAAGACGGCGATCGTGGTTCGCCGGGCGTTCCGGGTTCGCCGGGTCTGCCGGGTCCGGTCGGTCCGAAAGGTGAACCGGGGCCCACTGGTGCGCCAGGCTTGACCGGTCCGACCGGTGCGGTTGGCCTCCCGGGCCCACCGGGACCGAGCGGTCTGGTTGGCCCACAAGGTTCCCCGGGCTTACCGGGCCAGGTTGGAGAAACCGGTAAGCCGGGTGCACCGGGGCGCGACGGCGCAAGCGGTAAGGACGGCGACCGCGGTAGCCCGGGCGTGCCGGGTAGCCCGGGCCTGCCGGGCCCGGTGGGCCCCAAGGGTGAGCCGGGACCGACCGGCGCTCCGGGGTTGACCGGTCCAACGGGCGCTGTGGGCCTGCCGGGTCCACCGGGTCCGAGCGGTCTGGTTGGCCCGCAGGGTAGCCCGGGTCTGCCGGGCCAAGTTGGTGAAACCGGTAAACCGGGAGCACCAGGCCGTGATGGTGCCTCCGGTAAGGACGGCGATCGCGGTTCTCCGGGCGTCCCGGGCTCCCCGGGTCTGCCGGGCCCGGTGGGTCCGAAAGGTGAGCCGGGCCCGACGGGCGCGCCGGGCTTGACCGGCCCGACGGGTGCTGTGGGTCTGCCGGGCCCTCCGGGTCCAAGCGGTCTGGTGGGCCCTCAAGGTTCTCCGGGTCTGCCGGGACAGGTGGGCGAAACCGGTAAGCCGGGTGCGCCAGGTCGTGATGGCGCGAGCGGCAAAGATGGTGATCGTGGCAGTCCGGGGGTGCCGGGCAGCCCGGGCTTGCCGGGTCCAGTAGGTCCGAAAGGCGAGCCGGGCCCGACCGGCGCGCCT (SEQ ID NO: 8) and The nucleotide sequence of recombinant type VII humanized collagen C7P12 is GGAGAACCCGGGGCGAAGGGCGACCGCGGTCTGCCGGGTCCGCGTGGTGAAAAAGGTGAGGCGGGCCGCGCAGGCGAACCGGGTGACCCGGGCGAGGATGGTCAGAAAGGCGCGCCAGGTCCGAAAGGTTTTAAAGGCGATCCGGGCGAACCGGGTGCCAAGGGCGATAGAGGTCTGCCGGGTCCGCGTGGCGAAAAGGGTGAAGCGGGTCGTGCGGGTGAACCGGGTGACCCGGGCGAGGACGGTCAGAAGGGCGCGCCAGGTCCGAAAGGCTTCAAAGGTGACCCGGGTGAACCGGGCGCGAAAGGCGACCGTGGTTTACCGGGTCCGCGTGGTGAGAAGGGGGAGGCTGGTCGTGCCGGTGAACCGGGCGACCCAGGCGAGGATGGTCAGAAAGGCGCGCCTGGTCCCAAGGGCTTCAAGGGCGACCCGGGTGAACCGGGTGCCAAAGGGGATCGCGGTTTGCCAGGTCCTCGCGGTGAAAAGGGCGAGGCTGGTCGCGCTGGTGAGCCGGGCGACCCGGGTGAAGATGGTCAAAAAGGCGCTCCGGGTCCGAAGGGTTTTAAAGGTGATCCGGGCGAGCCGGGTGCGAAGGGCGATCGTGGCCTGCCGGGCCCACGTGGTGAGAAAGGCGAGGCCGGTCGTGCAGGCGAACCGGGTGACCCCGGCGAAGATGGCCAAAAGGGTGCGCCTGGCCCGAAGGGATTCAAAGGCGATCCGGGTGAGCCGGGCGCGAAAGGCGACCGCGGCCTGCCGGGTCCGCGTGGTGAGAAGGGCGAGGCAGGCCGTGCAGGTGAACCGGGTGACCCGGGTGAGGATGGTCAAAAAGGTGCTCCGGGTCCGAAGGGCTTTAAGGGCGACCCG (SEQ ID NO: 9) is as follows.

[0032] The recombinant humanized type VII collagen amino acid sequence produced in the present application is derived from the functional region of natural human type VII collagen, and includes proteins in which the functional region and similar functional regions and amino acid sequences have been mutated and modified, respectively.

[0033] The recombinant humanized type VII collagen produced in this application can be used in the production of high-end medical devices, such as bio-dressing materials, human biomimetic materials, cosmetic and plastic surgery materials, organoid culture materials, tissue injection and filling materials, skin repair and regeneration materials, oral mucosa repair and regeneration materials, cervical mucosa repair and regeneration materials, gynecological biomaterials, 3D printed artificial organ biomaterials, high-end cosmetic raw materials and high-end pharmaceutical auxiliary materials, and food additives.

[0034] Advantages of the present application include:

[0035] The present application provides the core functional region and amino acid sequence of recombinant humanized collagen VII, This application is the first to successfully synthesize recombinant humanized collagen type VII. The recombinant humanized type VII collagen produced in the present invention has a high cell adhesion effect or cell proliferation promoting effect, and does not induce an immune reaction when applied to the human body. The production method is simple, and recombinant humanized type VII collagen can be obtained on a large scale. Due to its excellent performance, recombinant humanized type VII collagen is expected to be widely used in the production of high-end cosmetic raw materials and pharmaceutical auxiliary materials, such as bio-dressing materials, human biomimetic materials, cosmetic and plastic surgery materials, organoid culture materials, tissue injection filling materials, skin repair and regeneration materials, oral mucosa repair and regeneration materials, cervical mucosa repair and regeneration materials, obstetrics and gynecology biomaterials, and 3D printing artificial organ biomaterials, as well as in the fields of food additives. [Brief explanation of the drawings]

[0036] [Figure 1]Purification status of recombinant type VII humanized collagen C7P7 (5: no sample was sampled, 6: the target protein was purified and collected, and the sampled flow-through sample was used). [Figure 2] 3 shows the purification status of recombinant humanized type VII collagen C7P11 (in 3 and 4, the target protein is the collected purified flow-through sample). [Figure 3] Purification status of recombinant humanized type VII collagen C7P12 (22 shows the purified and collected flow-through protein, 26 shows the protein after enzymatic cleavage, and 27 shows the protein after solution exchange). [Figure 4] FIG. 1 shows the growth-promoting effect of recombinant humanized type VII collagen C7P7. [Figure 5] FIG. 1 shows the growth-promoting effect of recombinant humanized type VII collagen C7P11. [Figure 6] FIG. 1 shows the growth-promoting effect of recombinant humanized type VII collagen C7P12. DETAILED DESCRIPTION OF THE INVENTION

[0037] In order to clarify the purpose, technical means and advantages of the present application, the technical means in the embodiments of the present application will be described below clearly and completely with reference to the embodiments of the present application, and it is obvious that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without any creative work fall within the scope of protection of the present application.

[0038] As used herein, a polypeptide is a plurality of amino acid residues linked via peptide bonds. As used herein, a polypeptide comprises a plurality of repeating units derived from human type VII collagen. A polypeptide comprises the amino acid sequence of SEQ ID NO. 1 (repeat unit). nHerein, the repeat units may be directly linked or may be linked via one or more amino acid residues. The number of repeat units, n, may be 4 to 20, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. The concentration of the polypeptide may be greater than 1 mg / ml, greater than 5 mg / ml, greater than 10 mg / ml, or greater than 12 mg / ml.

[0039] As used herein, the repeat unit of the present application may include or be the following sequence:

[0040] GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP(SEQ ID NO:1), GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAP (SEQ ID NO:2), or GEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDP(SEQ ID NO:3).

[0041] As used herein, a "nucleic acid" refers to a plurality of nucleotides linked together. The linkage between the nucleotides may be, for example, a phosphodiester bond. The nucleic acid herein may include a polynucleotide encoding a polypeptide of the present application. To facilitate subsequent processing of the polypeptide, the nucleic acid of the present application may further include nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag, and, if necessary, a nucleotide sequence encoding a leader sequence.

[0042] As used herein, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide can be inserted. A vector is called an expression vector if it can express a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, allowing the genetic material elements carried therein to be expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages, such as lambda phage or M13 phage, and animal viruses. A vector may contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication. A vector may contain the nucleic acid of the present application for introduction into a cell and expression. The vector may comprise expression control elements, such as a promoter, terminator and / or enhancer, operably linked to the nucleic acid.

[0043] As used herein, the term "host cell" refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA accelerated by electroporation, lipid transfection, and particle guns. Host cells may be eukaryotic or prokaryotic cells. For example, eukaryotic cells are yeast cells, animal cells, and / or insect cells. Prokaryotic cells may be E. coli cells.

[0044] As used herein, certain mutations may be present in the repeat units or polypeptides of the present application. For example, the amino acid sequence of one or more of these portions may include substitutions, deletions, additions, or insertions of amino acid residues. In other words, the present application allows for the use of repeat unit variants as long as they have the activity of promoting cell adhesion and / or proliferation. Specifically, the variants may have a certain percentage of identity with a specific sequence, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The specific sequence may be any sequence of the present application, such as SEQ ID NOs. 1 to 6, but preferably, these variants retain the core sequence identified by the present application.

[0045] The polypeptides of the present application may be produced in any suitable manner, for example, synthetically produced. Preferably, the polypeptides of the present application may be produced recombinantly.

[0046] The polypeptides, nucleic acids, vectors, and / or host cells of the present application may be prepared as compositions or kits. The compositions or kits may include one or more of the following: bio-covering materials, human biomimetic materials, cosmetic materials, organoid culture materials, cardiovascular stents, coating materials, tissue injection filling materials, ophthalmic materials, gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D-printed artificial organ biomaterials, cosmetic raw materials, medicinal supplements, and food additives. The compositions may be one or more of the following: bio-covering materials, human biomimetic materials, cosmetic materials, organoid culture materials, cardiovascular stents, coating materials, tissue injection filling materials, ophthalmic materials, gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D-printed artificial organ biomaterials, cosmetic raw materials, medicinal supplements, and food additives. The compositions or kits may promote cell adhesion or proliferation in vitro or promote cell adhesion or proliferation in vivo.

[0047] This application discloses specific screening and synthesis processes for the functional domain of recombinant humanized type VII collagen used in the production of human body structural materials. This application belongs to the field of synthetic biology. The biosynthetic production method for recombinant humanized type VII collagen in this application specifically includes the steps of (1) screening for the functional domain and constructing a bacterial strain, (2) fermenting, inducing, and expressing the organism, and (3) purifying and optionally enzymatically cleaving the humanized type VII collagen. The amino acid sequence of the recombinant humanized collagen produced in this application is derived from the functional domain of natural human type VII collagen, and includes proteins in which the functional domain and similar functional domains and amino acid sequences are mutated and modified, respectively. The recombinant humanized type VII collagen produced in this application has high activity in promoting cell adhesion and cell proliferation, does not induce immune reactions when applied to the human body, and its production method is novel, allowing for large-scale production of recombinant humanized type VII collagen, making it widely applicable to the production of human body structural materials. Its application areas include the production of high-end medical devices, such as bio-dressing materials, human biomimetic materials, cosmetic and plastic surgery materials, organoid culture materials, tissue injection filling materials, skin repair and regeneration materials, oral mucosa repair and regeneration materials, cervical mucosa repair and regeneration materials, obstetrics and gynecology biomaterials, 3D printed artificial organ biomaterials, high-end cosmetic raw materials and high-end pharmaceutical auxiliary materials, and food additives. [Example]

[0048] The present application is illustrated by the following examples. Those skilled in the art will understand that the examples are illustrative only and are not limiting. The present application is limited only by the scope of the appended claims.

[0049] Example 1 Construction and Expression of Recombinant Humanized Collagen VII A large-scale screening of functional regions was performed to obtain the following different recombinant humanized collagen VII target gene fragments:

[0050] Amino acid sequence of recombinant type VII humanized collagen C7P7 (SEQ ID NO:4): GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP, Amino acid sequence of recombinant type VII humanized collagen C7P11 (SEQ ID NO:5): GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAP GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAP GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAP GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAP Amino acid sequence of recombinant type VII humanized collagen C7P12 (SEQ ID NO:6): GEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDP (4) Base sequence of recombinant type VII humanized collagen C7P7 (SEQ ID NO:7): GGATTTCCCGGGGTCCCGGGAGGCACCGGCCCTAAAGGCGATCGTGGTGAAACCGGCAGCAAGGGCGAGCAGGGTCTGCCGGGCGAGCGCGGTTTGAGAGGCGAACCGGGTTTTCCAGGCGTGCCGGGCGGTACGGGTCCGAAGGGTGACCGTGGCGAAACCGGCAGCAAGGGTGAACAAGGTTTACCGGGTGAACGCGGTCTGCGTGGTGAGCCGGGCTTCCCAGGTGTTCCGGGCGGAACCGGTCCTAAAGGTGATCGTGGCGAAACCGGTTCCAAAGGCGAACAAGGTCTTCCGGGTGAGCGCGGTCTGCGTGGCGAACCGGGTTTCCCGGGCGTGCCGGGAGGCACCGGCCCAAAGGGCGACCGCGGAGAAACCGGCAGCAAAGGCGAGCAGGGCCTGCCGGGTGAACGTGGCCTGCGTGGTGAGCCGGGATTCCCGGGTGTTCCGGGCGGCACCGGTCCGAAAGGTGATCGTGGTGAAACCGGTAGCAAGGGTGAACAGGGTCTGCCGGGCGAGCGCGGCTTGAGAGGTGAGCCTGGTTTTCCGGGGGTGCCCGGCGGTACGGGCCCGAAAGGCGACCGTGGCGAAACCGGTTCTAAGGGTGAGCAGGGTCTGCCGGGTGAGCGTGGTCTGCGCGGTGAGCCGGGTTTCCCGGGCGTTCCGGGTGGCACTGGTCCGAAGGGCGACCGTGGCGAGACTGGCTCGAAAGGTGAACAGGGTTTGCCGGGTGAGCGTGGTCTGCGTGGTGAGCCGGGTTTTCCGGGCGTGCCGGGTGGCACGGGCCCAAAAGGCGATCGTGGTGAGACCGGTTCCAAGGGCGAGCAAGGTCTGCCGGGCGAGCGCGGTCTCCGCGGTGAACCG、 (5) The nucleotide sequence of recombinant humanized type VII collagen C7P11 (SEQ ID NO: 1)NO:8):GGACTAACAGGGCCGACCGGTGCGGTCGGCCTGCCGGGACCACCGGGCCCCAGCGGTCTGGTTGGTCCTCAGGGTTCCCCGGGTCTTCCGGGCCAGGTTGGTGAGACAGGCAAGCCGGGTGCGCCGGGCCGTGACGGTGCCTCTGGTAAAGACGGCGATCGTGGTTCGCCGGGCGTTCCGGGTTCGCCGGGTCTGCCGGGTCCGGTCGGTCCGAAAGGTGAACCGGGGCCCACTGGTGCGCCAGGCTTGACCGGTCCGACCGGTGCGGTTGGCCTCCCGGGCCCACCGGGACCGAGCGGTCTGGTTGGCCCACAAGGTTCCCCGGGCTTACCGGGCCAGGTTGGAGAAACCGGTAAGCCGGGTGCACCGGGGCGCGACGGCGCAAGCGGTAAGGACGGCGACCGCGGTAGCCCGGGCGTGCCGGGTAGCCCGGGCCTGCCGGGCCCGGTGGGCCCCAAGGGTGAGCCGGGACCGACCGGCGCTCCGGGGTTGACCGGTCCAACGGGCGCTGTGGGCCTGCCGGGTCCACCGGGTCCGAGCGGTCTGGTTGGCCCGCAGGGTAGCCCGGGTCTGCCGGGCCAAGTTGGTGAAACCGGTAAACCGGGAGCACCAGGCCGTGATGGTGCCTCCGGTAAGGACGGCGATCGCGGTTCTCCGGGCGTCCCGGGCTCCCCGGGTCTGCCGGGCCCGGTGGGTCCGAAAGGTGAGCCGGGCCCGACGGGCGCGCCGGGCTTGACCGGCCCGACGGGTGCTGTGGGTCTGCCGGGCCCTCCGGGTCCAAGCGGTCTGGTGGGCCCTCAAGGTTCTCCGGGTCTGCCGGGACAGGTGGGCGAAACCGGTAAGCCGGGTGCGCCAGGTCGTGATGGCGCGAGCGGCAAAGATGGTGATCGTGGCAGTCCGGGGGTGCCGGGCAGCCCGGGCTTGCCGGGTCCAGTAGGTCCGAAAGGCGAGCCGGGCCCGACCGGCGCGCCT、 (6) Nucleotide sequence of recombinant type VII humanized collagen C7P12 (SEQ ID NO:9):

[0051] The synthesized gene fragment was inserted into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid.

[0052] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process is as follows: (1) E. coli competent cells BL21(DE3) were removed from the ultra-low temperature refrigerator and placed on ice. When partially thawed, 2 μl of the target plasmid was added to the E. coli competent cells BL21(DE3) and mixed thoroughly 2-3 times. (2) The mixture was then placed on ice for 30 min, heat-shocked in a 42°C water bath for 45-90 s, removed, and placed on ice for 2 min. (3) The mixture was transferred to a biological safety cabinet, and 700 μl of liquid LB medium was added. The mixture was then cultured at 37°C and 220 rpm for 60 min. (4) 200 μl of the bacterial suspension was evenly spread on an LB plate containing kanamycin sulfate. (5) The plate was then cultured in a 37°C incubator for 15-17 h until uniformly sized colonies developed.

[0053] Five to six single colonies were selected from the transformed LB plate, placed in a shake flask containing an antibiotic (kanamycin sulfate) stock solution, and cultured for 7 hours at 220 rpm and 37°C in a constant temperature shaker. After culturing, the shake flask was cooled to 16°C, IPTG was added, and expression was induced for a certain period. After that, a sample was taken and subjected to electrophoresis detection ("Bacterial Solution" lane in Figures 1 to 3). The bacterial solution was then dispensed into a centrifuge flask and centrifuged at 8000 rpm at 4°C for 10 minutes. The bacterial cells were collected and their weight was recorded.

[0054] The collected bacterial cells were resuspended in the equilibrium working solution, the solution was cooled to below 15°C, homogenized, and homogenized twice under high pressure. After completion, the bacterial solution was collected ("Homogenization" lanes in Figures 1-3). The homogenized bacterial solution was dispensed into centrifuge flasks and centrifuged at 17,000 rpm at 4°C for 30 minutes. The supernatant was collected, and the supernatant and precipitate were subjected to electrophoresis detection ("Supernatant" and "Precipitate" lanes in Figures 1-3).

[0055] Recombinant humanized type VII collagen was purified and enzymatically cleaved. The specific process was as follows: (1) For crude purification, a) the column was washed with water. b) the column was equilibrated with an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). c) For sampling, the supernatant after centrifugation was added to the column until the liquid stopped flowing, and the flow-through liquid was subjected to electrophoretic detection ("Flow-through" lanes in Figures 1 to 3). d) For washing the hybrid protein, 25 mL of scrubbing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added until the liquid stopped flowing, and the flow-through liquid was subjected to electrophoretic detection ("Scrubbing" lanes in Figures 1 to 3). e) For collection of the target protein, 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) was added, and the flow-through solution was collected. The protein concentration was measured by UV-visible spectrophotometry. The protein concentration was calculated according to the following formula: C (mg / mL) = A280 x dilution factor x extinction coefficient. Electrophoretic detection was performed ("Elution" lanes in Figures 1-3). f) The column was washed with 1 M imidazole working solution ("1 M Wash" lanes in Figures 1-3). g) The column was washed with purified water. (2) For enzymatic cleavage ("After Cleavage" lanes in Figures 1-3), TEV enzyme was added so that the ratio of total protein to total TEV enzyme was 20:1. Enzymatic cleavage was performed at 16 °C for 2 h to obtain the target collagen. Samples were then sampled and electrophoretically detected. The protein solution after enzymatic cleavage was placed in a dialysis bag and dialyzed at 4°C for 2 hours, then transferred to fresh dialysate and dialyzed overnight at 4°C. (3) Regarding precision purification, a) column equilibration was performed using solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 ml / min. b) sampling was performed at a flow rate of 5 ml / min, and the flow-through solution was collected to obtain recombinant humanized type VII collagen C7P7, C7P11, and C7P12, which were then subjected to electrophoretic detection. Lane 6 in Figure 1 shows the apparent molecular weight of recombinant humanized type VII collagen C7P7, which closely matches the predicted molecular weight of 27.8 kDa.Lanes 3 and 4 in Figure 2 show the apparent molecular weight of recombinant humanized type VII collagen C7P11, which is approximately consistent with the predicted molecular weight of 28.5 kDa. Lane 26 in Figure 3 shows the apparent molecular weight of recombinant humanized type VII collagen C7P12, which is approximately consistent with the predicted molecular weight of 27.5 kDa. To further confirm the identities of the separated C7P7, C7P11, and C7P12, the inventors performed mass spectrometry detection of the recombinant humanized type VII collagen in Example 3.

[0056] Example 2: Detection of the cell proliferation-promoting activity of recombinant humanized type VII collagen For the method of detecting collagen's cell proliferation promoting activity, reference may be made to the People's Republic of China Pharmaceutical Industry Standard YYT1849-2022 Recombinant Collagen. The specific implementation method is as follows:

[0057] (1) The principle of this method is as follows: The CCK8 reagent contains WST8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-benzenedisulfonic acid)-2H-tetrazole monosodium salt], which is reduced to a highly water-soluble yellow formazan dye by dehydrogenase in cells through the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-methoxy PMS). The number of formazan products produced is proportional to the number of viable cells. Therefore, this property can be used to directly analyze cell proliferation and toxicity.

[0058] 2) Regarding the grouping of the test, complete medium DMEM was the negative control, 8% NaCl was the positive control, collagen solution (solution in 8% NaCl) was the test sample, and complete medium without cells was the zero adjustment well.

[0059] (3) Regarding sample preparation, collagen was dissolved in complete medium to the maximum concentration, filtered through a 0.22 μm microfiltration membrane to sterilize, and prepared at each concentration. NaCl was dissolved in complete medium to a final concentration of 8%, and then filtered to sterilize.

[0060] (4) Regarding cell seeding in plates, when the NIH 3T3 cells were about 90% confluent, they were trypsinized and counted. Each well contained (5-10) x 10 3 Cells were seeded into a 96-well plate, and 100 μL of PBS was added to the peripheral wells to block the cells. The cells were then cultured for 24 hours to allow adhesion.

[0061] (5) Regarding cell administration, after cell adhesion, the supernatant was aspirated and discarded, and the solution of the corresponding group was replaced in turn. Each group had four duplicate wells, and the cells were cultured for 48 hours.

[0062] (6) For experimental detection, the culture medium was discarded, 100 μL of basal medium (containing 5 μL of CCK8 solution) was added, and the cells were incubated in an incubator for 1-2 h. Then, detection was performed using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 450 nm. The calculation formula for cell viability was as follows:

[0063] Cell viability = {(As - Ab) / (Ac - Ab)} x 100%, where As is the absorbance of the recombinant collagen test well, Ab is the absorbance of the zero-adjusted well, and Ac is the absorbance of the negative well.

[0064] Figure 4 shows the proliferation-promoting effect of recombinant type VII humanized collagen C7P7. Figure 5 shows the proliferation-promoting effect of recombinant type VII humanized collagen C7P11. Figure 6 shows the proliferation-promoting effect of recombinant type VII humanized collagen C7P12. As shown in Figures 4 to 6, the cell viability of the positive control group was zero compared to the negative control group, and the difference is statistically significant. C7P7 showed a significant proliferation-promoting effect at 12 mg / ml, while C7P11 and C7P12 did not promote cell proliferation. Unexpectedly, the inventors discovered that C7P7 can promote cell proliferation compared to C7P11 and C7P12, as evidenced by increased cell viability (see Table 1).

[0065] Table 1 Cell viability [Table 1]

[0066] Example 3 Mass Spectrometric Detection of Recombinant Humanized Collagen Type VII Experimental Method [Table 2]

[0067] Protein samples were reduced with DTT, alkylated with iodoacetamide, and then enzymatically digested overnight with trypsin. The resulting peptide fragments were desalted using a C18 ZipTip, mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA), and plated. Finally, the samples were analyzed using a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF / TOF) Ulraflextreme mass spectrometer. TM , Brucker, Germany (for peptide mass fingerprinting techniques see Protein J. 2016;35:212-7).

[0068] Data searches are performed from the MS / MS Ion Search page on the local masco site. Protein identification results are obtained from the primary mass spectra of peptide fragments generated after enzymatic digestion. For detection parameters, two uncleaved sites are set for trypsin enzymatic digestion. Cysteine alkylation is set as a fixed modification. Methionine oxidation is set as a variable modification. The database used for identification is NCBprot.

[0069] Table 2. C7P7 mass spectrometry detected molecular weights and corresponding polypeptides [Table 3] GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEPGFPGVPGGTGPKGDRGETGSKGEQGLPGERGLRGEP GFPGVPGGTGPKGDRGETGSKGEQGLPGER GLRGEP (SEQ ID NO: 21) The coverage rate of the detected polypeptide fragments compared with the theoretical sequence was 97.91%, making the detection results highly reliable.

[0070] Table 3. C7P11 mass spectrometry detected molecular weight and corresponding polypeptide [Table 4] GLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGL TGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAPGLTGPTGAVGLPGPPGPSGLVGPQGSPGLPGQVGETGKPGAPGRDGASGKDGDRGSPGVPGSPGLPGPVGPKGEPGPTGAP(SEQ ID NO: 28)

[0071] The detected polypeptide fragments have a coverage rate of 100% compared with the theoretical sequence, making the detection results highly reliable.

[0072] Table 4. C7P12 mass spectrometry detected molecular weight and corresponding polypeptide [Table 5] GEPGAK GDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRA GEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPKGFKGDPGEPGAKGDRGLPGPRGEKGEAGRAGEPGDPGEDGQKGAPGPK GFKGDP GEKGEAGRAGEPGDPGEDGQKGAPGPK GFKGDP (SEQ ID NO: 35)

[0073] The coverage rate of the detected polypeptide fragments compared with the theoretical sequence was 93.75%, making the detection results very reliable.

[0074] The above examples are preferred embodiments of the present application, and the embodiments of the present application are not limited to the above examples. Any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present application are all equivalent substitutions and fall within the scope of protection of the present application.

Claims

1. (Repeating unit) ｎ wherein the repeat units comprise the amino acid sequence of SEQ ID NO. 1, wherein each repeat unit is directly linked, and the number of repeat units n is 4 to 20, and preferably said polypeptide is recombinant humanized collagen VII.

2. 2. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO.

4.

3. 3. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to claim 1 or 2, optionally further comprising a nucleotide sequence encoding a purification tag such as a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, and optionally further comprising a nucleotide sequence encoding a leader sequence.

4. 4. The nucleic acid of claim 3, comprising the nucleotide sequence of SEQ ID NO.

7.

5. A vector comprising a nucleic acid according to claim 3 or 4, and optionally comprising expression control elements, such as a promoter, terminator and / or enhancer, operably linked to said nucleic acid.

6. 10. A host cell comprising a nucleic acid according to claim 3 or 4 or a vector according to claim 5, which is preferably a eukaryotic or prokaryotic cell, preferably wherein the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or wherein the prokaryotic cell is an E. coli cell, such as E. coli BL21.

7. (1) culturing the host cell of claim 6 under suitable culture conditions; (2) harvesting the host cells and / or medium containing the polypeptide; For example, a method for producing the polypeptide described in claim 1 or 2, comprising: (1) a step of crudely purifying the polypeptide using a Ni affinity chromatography column; (2) a step of adding TEV enzyme to perform enzymatic cleavage; and (3) a step of purifying the fusion protein, comprising a step of finely purifying the polypeptide using an ion exchange column.

8. A composition comprising the polypeptide of claim 1 or 2, the nucleic acid of claim 3 or 4, the vector of claim 5 and / or the host cell of claim 6, and preferably being one or more of a biological covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biological material, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a medicinal auxiliary material and a food additive.

9. Use of a polypeptide according to claim 1 or 2, a nucleic acid according to claim 3 or 4, a vector according to claim 5, a host cell according to claim 6 and / or a composition according to claim 8 in promoting cell adhesion or cell proliferation in vitro or in the manufacture of a product or kit for promoting cell adhesion or cell proliferation.

10. The application of the polypeptide according to claim 1 or 2, the nucleic acid according to claim 3 or 4, the vector according to claim 5, the host cell according to claim 6 and / or the composition according to claim 8 in the production of high-end medical devices, such as bio-coating materials, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stents, coating materials, tissue injection filling materials, ophthalmic materials, gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, high-end cosmetic raw materials and medicinal auxiliary materials, and food additives.

11. 10. A method for promoting cell proliferation, comprising contacting a cell with a polypeptide according to claim 1 or 2, wherein the cell is preferably an animal cell, such as a mammalian cell.

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