WO2024016740A1 - 一种生物合成人体结构性材料的制备方法 - Google Patents

一种生物合成人体结构性材料的制备方法 Download PDF

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WO2024016740A1
WO2024016740A1 PCT/CN2023/087212 CN2023087212W WO2024016740A1 WO 2024016740 A1 WO2024016740 A1 WO 2024016740A1 CN 2023087212 W CN2023087212 W CN 2023087212W WO 2024016740 A1 WO2024016740 A1 WO 2024016740A1
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amino acid
polypeptide
materials
seq
repeating unit
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PCT/CN2023/087212
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English (en)
French (fr)
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杨霞
张兴栋
王云兵
杨立
何振瑞
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山西锦波生物医药股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A61L15/325—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • This application belongs to the field of synthetic biology technology and relates to a biosynthetic preparation method of human structural materials.
  • Structural materials of the human body are mainly structural proteins including collagen. These proteins have adhesion and support functions for cells and tissues and are the main components of the extracellular matrix. Collagen is a type of protein widely distributed in human connective tissue. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Currently, it is found that there are at least 28 collagen subtypes in the human body, which are located in different tissues. organ. Among them, type V collagen is a fibrillar collagen, which is often expressed along with type I collagen, but the content is small.
  • type V collagen has a low content in the body, is a minor collagen, and the extraction process is complicated.
  • animal-derived immune responses are also an important reason for the limited application of collagen.
  • biosynthetic pathways to obtain collagen has become increasingly mature, especially humanized collagen, which has been at the forefront of the world.
  • the State Food and Drug Administration made a naming and classification of biosynthetic collagen.
  • recombinant humanized collagen refers to the full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombinant technology, or It is a combination containing functional fragments of human collagen.
  • Type V collagen was first discovered in human placenta and skin. It contains three ⁇ chains with different strengths and mobility, and will form four different subtypes in tissues, namely ⁇ 1 ⁇ 1 ⁇ 2(V), ⁇ 1 ⁇ 2 ⁇ 3(V), and ⁇ 3 ⁇ 3 ⁇ 3 (V), a mixture of type V collagen ⁇ chain and type XI collagen ⁇ chain. three different alpha chains Procollagen molecules, composed of three rope-like strands, are assembled into slender collagen fibers after extracellular enzymatic digestion.
  • the slender fibers are cross-linked with each other in the space around the cells, and after cross-linking, they form a high-strength mature type V collagen fiber network; or they form heterotrimers with other types of collagen such as type XI collagen and are distributed in the cornea, Skin, ligaments, bones, tendons, muscles and other parts.
  • Type V collagen has the common structural characteristics of fibrillar collagen, with an uninterrupted sequence of more than 1,000 glycine-Xaa-Yaa in the central region, where Xaa-Yaa is any amino acid or imino acid residue. Structurally speaking, the structure of the human body's natural type V collagen is very complex, which makes humanized collagen difficult to express and prepare in large quantities through conventional means.
  • type V collagen mainly involves processing animal-derived tissues with pepsin to extract collagen derivatives.
  • the collagen extracted by these methods has lost its original biological activity and cannot exert its true function.
  • people have been able to prepare recombinant humanized collagen in animal, plant, and microbial expression systems through transgenic technology, solving the shortcomings of enzymatic hydrolysis.
  • recombinant type V humanized collagen has not been successfully prepared. Therefore, there is an urgent need for a biosynthetic method for recombinant type V humanized collagen so that it can be widely used as a structural material for the human body.
  • the inventor designed for the first time a functional region screening and protein synthesis process for recombinant type V humanized collagen.
  • the inventor found the amino acid sequence of the core region of type V collagen (SEQ ID NO. 1, gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv), and used the core region as a repeating unit to construct a polypeptide containing multiple repeating units.
  • the inventor found that the constructed polypeptide has the activity of promoting cell adhesion.
  • the inventor also found that one or more amino acids can be extended as repeating units at the N-terminus and/or C-terminus of the core region, and the constructed polypeptide containing multiple repeating units also has the activity of promoting cell adhesion.
  • the polypeptide may also include a peptide segment of a certain length outside the repeating units (for example, the N-terminus and/or C-terminus of the polypeptide composed of the repeating units).
  • the peptide segment may be a continuous amino acid segment starting from position 1 of the repeating unit.
  • the application provides a polypeptide comprising (repeating unit) n or (repeating unit) n - the structure of the C-terminal region, the repeating unit comprising the amino acid sequence of SEQ ID NO. 1, or the repeating unit comprising SEQ ID NO. .1 and additional amino acid residues at the N-terminus and/or C-terminus of SEQ ID NO.1, the number of the additional amino acid residues is 1-50.
  • the polypeptide of the present application is recombinant type V humanized collagen.
  • each repeating unit is directly linked and the number n of repeating units is 4-20, and the C-terminal region is a continuous amino acid stretch starting from position 1 of the repeating unit.
  • the additional amino acid residue is at the C-terminus of SEQ ID NO. 1 and is the amino acid sequence (galglk) shown in SEQ ID NO. 7 or a continuous amino acid segment of the amino acid sequence.
  • the additional amino acid residue is a continuous amino acid segment starting from position 1 of the amino acid sequence shown in SEQ ID NO. 7.
  • the repeating units comprise the amino acid sequence of any one of SEQ ID Nos. 2, 3 and 8-11.
  • the polypeptide comprises an amino acid sequence selected from SEQ ID NO. 4-6.
  • the application provides a nucleic acid comprising a nucleotide of a polypeptide of the application.
  • the nucleic acid further comprises nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag.
  • a purification tag such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag.
  • the nucleic acid further comprises nucleotides encoding a leader sequence.
  • the application provides a vector comprising a nucleic acid according to the application.
  • the vector contains expression control elements, such as promoters, terminators, and/or enhancers, operably linked to the nucleic acid.
  • the present application provides a host cell comprising a nucleic acid or vector of the present application.
  • the host cell is a eukaryotic or prokaryotic cell.
  • the eukaryotic cell is a yeast cell, an animal cell, and/or an insect cell.
  • the prokaryotic cell is an E. coli cell.
  • the application provides for the production of a polypeptide of the application, comprising:
  • compositions comprising the polypeptides, nucleic acids, vectors and/or host cells of the present application.
  • the composition is a biological dressing, human bionic material, plastic surgery material, organoid culture material, cardiovascular stent material, coating material, tissue injection filling material, ophthalmic material, obstetrics and gynecology biomaterial, nerve repair One or more of regenerative materials, liver tissue materials and blood vessel repair regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.
  • the application provides the polypeptides, nucleic acids, vectors, host cells and /or the use of the composition to promote cell adhesion in vitro or in the preparation of a medicament that promotes cell adhesion.
  • the present application provides the polypeptides, nucleic acids, vectors, host cells and/or compositions of the present application for use in the preparation of high-end medical devices, such as biological dressings, human bionic materials, plastic and cosmetic materials, organoid culture, and cardiovascular stents. , coating, tissue injection filling, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, 3D printing artificial organ biomaterials, etc.; high-end cosmetic raw materials and high-end pharmaceutical excipients; applications in food additives .
  • high-end medical devices such as biological dressings, human bionic materials, plastic and cosmetic materials, organoid culture, and cardiovascular stents.
  • coating tissue injection filling, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, 3D printing artificial organ biomaterials, etc.
  • This application discloses a specific process of functional area screening and synthesis process of recombinant type V humanized collagen, which can be used to prepare human body structural materials.
  • This application belongs to the field of synthetic biology technology.
  • This application discloses a specific process of functional area screening and synthesis process of recombinant type V humanized collagen, which can be used to prepare human body structural materials.
  • This application belongs to the field of synthetic biology technology.
  • the biosynthetic method for preparing recombinant type V humanized collagen in this application includes: (1) functional area screening and strain construction; (2) biological fermentation, induction and expression; (3) humanized V Purification and optional enzymatic digestion of collagen.
  • the amino acid sequence of the recombinant humanized collagen prepared in this application is derived from the functional region of human natural type V collagen, and includes this functional region and similar functional regions, as well as proteins with mutated and modified amino acid sequences.
  • the recombinant type V humanized collagen prepared by this application has high activity in promoting cell adhesion and will not produce an immune response when applied to the human body. Moreover, its preparation method is novel, and the recombinant type V humanized collagen can be obtained on a large scale. It has been widely used in the preparation of human body structural materials.
  • high-end medical device preparation such as biological dressings, human bionic materials, plastic surgery materials, organoid culture, cardiovascular stents, coatings, tissue injection filling, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration, liver Tissue and blood vessel repair and regeneration, 3D printing of artificial organ biomaterials, etc.; high-end cosmetic raw materials and high-end pharmaceutical excipients; food additives, etc.
  • this application provides a method for biosynthesizing recombinant type V humanized collagen, that is, a method for preparing human structural materials.
  • the specific process includes: (1) Functional area screening, bacterial strain selection Construction; (2) large-scale biological fermentation culture and protein induction expression; (3) purification and optional enzyme digestion of humanized type V collagen.
  • functional area screening and strain construction can be carried out as follows: (1) Large-scale functional area screening to obtain the target gene functional area; (2) Insert the obtained target gene functional area into pET-32a for expression The recombinant expression plasmid is obtained from the vector; (3) The recombinant expression plasmid is transferred into Escherichia coli competent cells BL21 (DE3), and positive Escherichia coli genetically engineered bacteria are screened.
  • large-scale biological fermentation can be carried out as follows: the positive large intestine obtained by screening Bacillus genetically engineered bacteria were added to the shake flask of the antibiotic stock solution, and cultured in a constant-temperature shaker at 220 rpm and 37°C for 7 h.
  • the induced expression of the protein can be carried out as follows: (1) Cool the cultured shake flask to 16°C; (2) Add IPTG stock solution for induced expression; (3) Add the induced expression bacteria to The liquid was placed in a centrifuge bottle, and centrifuged at 8000 rpm and 4°C for 10 min to collect the bacteria.
  • the purification and optional enzymatic digestion of humanized type V collagen can be carried out as follows: (1) crudely purifying humanized type V collagen on a Ni affinity chromatography column; (2) Add TEV enzyme digestion according to a certain proportion; (3) Ion exchange column to purify humanized type V collagen.
  • the screened functional region is as follows: (1) C5V3G1 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglggkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpg
  • the amino acid sequence of the recombinant type V humanized collagen prepared in this application is derived from the functional region of human natural type V collagen, and includes this functional region and similar functional regions, as well as proteins with mutated and modified amino acid sequences.
  • the application fields of the recombinant type V humanized collagen prepared in this application include the preparation of high-end medical devices, such as biological dressings, human bionic materials, plastic surgery materials, organoid culture, cardiovascular stents, coatings, and myocardial repair. , tissue injection filling, ophthalmic materials, obstetrics and gynecology biological materials, Nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, 3D printed artificial organ biomaterials, tumor prevention, bone repair materials, skin repair materials, kidney tissue repair, pancreas repair, etc.; high-end cosmetic raw materials and high-end pharmaceutical excipients; food additives, etc.
  • high-end medical devices such as biological dressings, human bionic materials, plastic surgery materials, organoid culture, cardiovascular stents, coatings, and myocardial repair.
  • This application provides the core functional region and amino acid sequence of recombinant type V humanized collagen
  • the recombinant type V humanized collagen prepared by this application has good cell adhesion effect and will not produce an immune response when applied to the human body;
  • the preparation method is simple and recombinant type V humanized collagen can be obtained on a large scale.
  • FIG. 1 Electrophoresis detection chart of recombinant type V humanized collagen C5V3G1 and C5V3G2 after crude purification. Electropherograms after each purification step are shown.
  • FIG. 2 Electrophoresis detection chart of recombinant type V humanized collagen C5V3G3 after crude purification and electrophoresis detection chart of C5V3G1, C5V3G2, and C5V3G3 after enzyme digestion. Electropherograms after each purification step are shown.
  • FIG. 3 Electrophoresis detection chart of recombinant type V humanized collagen C5V3G1, C5V3G2, and C5V3G3 after purification.
  • Figure 4 Cell adhesion activity of recombinant type V humanized collagen C5V3G1.
  • Figure 5 Cell adhesion activity of recombinant type V humanized collagen C5V3G2.
  • Figure 6 Cell adhesion activity of recombinant type V humanized collagen C5V3G3.
  • a polypeptide refers to a plurality of amino acid residues linked by peptide bonds.
  • a polypeptide contains multiple repeating units derived from human type V collagen (GenBank: KAI4009058.1).
  • the polypeptide may comprise a structure containing (repeating unit) n or (repeating unit) n -C-terminal region, the repeating unit comprising the amino acid sequence of SEQ ID NO. 1, or the repeating unit comprising SEQ ID NO. 1 and additional amino acid residues at the N-terminus and/or C-terminus of SEQ ID NO. 1, and the number of the additional amino acid residues is 1-50.
  • the number of additional amino acid residues is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49.
  • the additional amino acid residues may be derived from human type V collagen.
  • the additional amino acid residues may be consecutive amino acid residues directly adjacent to the repeating unit in situ in native human type V collagen.
  • the additional amino acid residue may be the amino acid sequence shown in SEQ ID NO. 7 or a continuous amino acid segment of the amino acid sequence.
  • the additional amino acid residue may be a continuous amino acid segment starting from position 1 of the amino acid sequence shown in SEQ ID NO.7.
  • the additional amino acid residue may be located at the C-terminus.
  • each repeating unit may be directly linked or may be separated by one or more amino acid residues.
  • the number n of repeating units may be 4-20, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.
  • the polypeptide may contain a C-terminal region.
  • the C-terminal region may be a contiguous amino acid stretch starting from position 1 of the repeating unit.
  • the repeating unit may comprise the amino acid sequence of any one of SEQ ID NO. 2 or 3.
  • section when used in connection with amino acids refers to a portion of a sequence that is smaller than the specified sequence.
  • amino acid stretch in SEQ ID NO.7 refers to a partial sequence of any length less than the length of SEQ ID NO.7.
  • continuous stretch of amino acids refers to a stretch of amino acids consisting of directly adjacent amino acids in a specified sequence.
  • C-terminus and N-terminus refer to the position relative to the polypeptide or specific amino acid sequence, specifically the carboxyl or amino terminus of the polypeptide or specific amino acid sequence. direction.
  • the position is described relative to the C-terminus of the polypeptide or specific amino acid sequence.
  • the amino acid at position 1 refers to G.
  • the repeating units of the present application may include or may be the following sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv(SEQ ID NO.1); gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal(SEQ ID NO.2); gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk(SEQ ID NO.3); gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvg(SEQ ID NO.8); gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvga (SEQ ID NO.9); gkegtkgdpgpaglpgkdgppglrgfpgdrglp
  • nucleic acid refers to a plurality of nucleotides linked by internucleotides.
  • the internucleotide linkage may be, for example, a phosphodiester bond.
  • Nucleic acids herein may comprise polynucleotides encoding polypeptides of the present application.
  • the nucleic acid of the present application can also include nucleotides encoding purification tags, such as His tag, GST tag, MBP tag, SUMO tag or NusA tag, and nucleotides encoding the leader sequence when necessary.
  • the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Vectors can contain a variety of expression control elements, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain an origin of replication site.
  • Vectors may contain nucleic acids of the present application for easy introduction into cells for expression.
  • the vector may contain expression control elements, such as promoters, terminators and/or enhancers, operably linked to the nucleic acid.
  • the term "host cell” is a cell into which nucleic acid molecules have been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipofection, and particle guns.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Eukaryotic cells are, for example, yeast cells, animal cells and/or insect cells.
  • the prokaryotic cell can be an E. coli cell.
  • certain mutations may exist in various parts of the polypeptide of the present application, such as the repeating unit or the C-terminal region.
  • the amino acid sequence of one or more of these portions may contain substitutions, deletions, additions or insertions of amino acid residues. That is, variants of various portions, such as repeat unit variants or C-terminal region variants, may be used in the present application, as long as the variant retains the activity of promoting cell adhesion.
  • a variant can have a certain percent identity to a specified sequence, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identity.
  • the specified sequence can be any sequence of the present application, such as SEQ ID NO. 1-6, but it is preferred that these variants retain the core sequence identified in the present application.
  • the polypeptides of the present application can be prepared by any suitable means, for example, by synthesis.
  • the polypeptide of the present application can be produced recombinantly.
  • the method may include any one or more of the following steps: (1) cultivating the host cell according to claim 8 under suitable culture conditions; (2) harvesting the host cell and/or culture medium containing the polypeptide ; and (3) purifying the fusion protein or trimeric fusion protein.
  • the coding sequence of the polypeptide of the present application can be inserted into a suitable expression vector, such as PET-28a, to obtain a recombinant expression vector.
  • the recombinant expression plasmid can be transferred into E. coli competent cells BL21 (DE3), and positive E.
  • E. coli genetically engineered bacteria can be obtained by screening. Then, the screened positive E. coli genetically engineered bacteria can be subjected to large-scale biological fermentation (for example, cultured in a constant temperature shaker at 220 rpm and 37°C for 7 hours). When cultured to the appropriate cell density, cells can be induced to express proteins. For example, in the case of E. coli genetically engineered bacteria, (1) cool the cultured shake flask to 16°C; (2) add IPTG stock solution to induce expression; (3) place the induced expression bacterial solution in a centrifuge In the bottle, centrifuge at 8000 rpm and 4°C for 10 min to collect the bacteria.
  • the bacterial cells can be lysed (for example, through high-pressure homogenization) to obtain bacterial cell lysate, and then centrifuged to obtain a supernatant.
  • the supernatant can be purified to obtain purified polypeptide.
  • the purification process may include one or more of the following steps: (1) crudely purifying the polypeptide on a Ni affinity chromatography column; (2) adding TEV enzyme digestion in a certain proportion; and (3) purifying the polypeptide on an ion exchange column.
  • compositions or kits can be prepared into compositions or kits.
  • the composition or kit may include biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, and nerve repair and regeneration materials. , liver tissue materials and blood vessel repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.
  • the composition can be biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue Materials and one or more of vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.
  • the composition or kit can be used to promote cell adhesion in vitro or to promote cell adhesion in vivo.
  • Example 1 Construction and expression of recombinant type V humanized collagen
  • gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk (C5V3G1 amino acid sequence: SEQ ID NO.6; repeat sequence SEQ ID NO.3, gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk); (2) C5V3G2 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglp
  • gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal C5V3G2 amino acid sequence SEQ ID NO: 5, repeating unit SEQ ID NO. 2, gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal); (3) C5V3G3 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrg lpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfp
  • gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv C5V3G3 amino acid sequence: SEQ ID NO: 4,
  • the repeating unit is SEQ ID NO. 1: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv).
  • C5V3G1 base sequence SEQ ID NO: 12
  • C5V3G2 base sequence SEQ ID NO: 13
  • C5V3G3 base sequence GGGAAAGAAGGAACGAAAGGCGATCCGGGTCCGGCGGGCCTGCCGGGTAAGGACGGCCCGCCGGGATTACGCGGTTTCCGGGTGACCGCGGTTTGCCGGGTCCGGTTGGCAAAGAAGGTACTAAGGGTGATCCGGGCCCAGCC GGTCTGCCGGGCAAGGATGGTCCGCCAGGCCTGAGAGGCTTCCCGGGTGACCGCGGGCTGCCGGGCCCGGTTGGTAAGGAGGGCACCAAAGGCGATCCGGGTCCGGCGGGTTTGCCGGGCAAGGATGGCCCGCCAGGCCTGCGTGGCTTTCCGGGTGATCGTGGTCTGCCGGGCCCCGTGGGCAAAGAGGGCACCAAAGGTGATCCGGGTCCAGCGGGTCTGCCAGGTAAGGACGGTCCGCCTGGCCTGCGTGGT TTCCCGGGT
  • Collect the target protein add 20mL of eluate and collect the flow-through to obtain the target protein containing His tag.
  • Use UV-visible spectrophotometry to detect the protein concentration according to the following formula (C (mg/ml) A280 ⁇ dilution factor ⁇ extinction coefficient) to calculate the protein concentration and conduct electrophoresis detection (see Figure 1 and Figure 2, "Elution" lane).
  • C (mg/ml) A280 ⁇ dilution factor ⁇ extinction coefficient) to calculate the protein concentration and conduct electrophoresis detection (see Figure 1 and Figure 2, "Elution" lane).
  • Enzyme digestion The ratio of the total amount of protein to the total amount of TEV enzyme is 20:1, add TEV enzyme, digest at 16°C for 2 hours, and take samples for electrophoresis detection.
  • the principle of this method is to measure the characteristic absorption of peptide bonds under far ultraviolet light, which is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for the detection of human collagen and its analogues that do not develop color in Coomassie brilliant blue. . (The reference is Walker JM.The Protein Protocols Handbook, second edition.HumanaPress.43-45.). After detecting the protein concentration, use PBS to adjust the concentration of all proteins to be tested to 0.25 mg/mL, 0.5 mg/mL, or 1 mg/ml.
  • the cell adhesion rate (relative cell adhesion activity) can reflect the activity of collagen. The higher the activity of the protein, the better it can provide cells with a high-quality external environment in a short time and help cells adhere to the wall.
  • Figures 4, 5, and 6 The results are shown in Figures 4, 5, and 6. From the comparison, it can be seen that compared with bovine type I collagen (B ColI group, 0.5 mg/ml), the recombinant type V humanized collagen of the present application has better Bioadhesive activity.
  • Figure 4 depicts the relative cell adhesion activity of C5V3G1 relative to bovine type I collagen, indicating that C5V3G1 has cell adhesion activity and has higher cell adhesion activity than bovine type I collagen at concentrations of 0.25, 0.5 and 1 mg/ml.
  • Figure 5 shows the relative cell adhesion activity of C5V3G2 relative to bovine type I collagen, indicating that C5V3G2 has better cell adhesion activity at concentrations above 0.25 mg/ml.
  • Figure 6 shows the relative cell adhesion activity of C5V3G3 relative to bovine type I collagen, indicating that C5V3G3 has cell adhesion activity and has higher cell adhesion activity than bovine type I collagen at a concentration of 0.5 mg/ml.
  • Example 3 Mass spectrometry detection of recombinant type V humanized collagen
  • the protein identification results are obtained based on the primary mass spectrometry of the peptide fragments produced after enzymatic digestion. Detection parameters: Trypsin enzymatic hydrolysis, with two missed cleavage sites. Set alkylation of cysteine as a fixed modification. Oxidation of methionine is a variable modification.
  • the database used for identification is NCBprot.
  • the coverage rate of the detected polypeptide fragments was 100%, which was consistent with the theoretical sequence, and the detection results were very credible.
  • the coverage rate of the detected polypeptide fragments was 83%, and the detection results were very credible. .
  • the coverage rate of the detected polypeptide fragments was 96.11%, and the detection results were very credible.

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Abstract

提供了一种生物合成人体结构性材料的制备方法。本申请的多肽，其包含(重复单元)n或(重复单元)n-C端区的结构，该重复单元包含SEQ ID NO.1的氨基酸序列。本申请制备得到的重组Ⅴ型人源化胶原蛋白具有高促进细胞黏附的活性，应用于人体不会产生免疫反应，且其制备方法新颖，可大规模获得重组Ⅴ型人源化胶原蛋白，在人体结构性材料制备方面得到广泛应用。其应用领域包含高端医疗器械制备，如生物敷料、人体仿生材料、整形美容材料、类器官培养、心血管支架、涂层、组织注射填充、眼科材料、妇产科生物材料、神经修复再生、肝脏组织及血管修复再生、3D打印人造器官生物材料等。

Description

一种生物合成人体结构性材料的制备方法
本申请要求申请日为2022年7月19日，申请号：202210849498.3，发明名称为“一种生物合成人体结构性材料的制备方法”的中国发明专利申请的优先权权益，其通过引用并入本文。
技术领域
本申请属于合成生物技术领域，涉及一种人体结构性材料的生物合成制备方法。
背景技术
人体结构性材料主要是包含胶原蛋白在内的结构性蛋白，这类蛋白对细胞、组织具有黏附、支撑功能，是主要的细胞外基质组成成分。胶原蛋白是广泛分布于人体结缔组织的一类蛋白质，也是人体含量最多的蛋白质，可以占到蛋白质总量的25％～35％，目前发现人体至少有28种胶原蛋白亚型，分别位于不同组织器官。其中Ⅴ型胶原蛋白属于纤维状胶原蛋白，常常伴随着Ⅰ型胶原蛋白表达，但含量较少，具有独特的生理功能，如：抑制表皮、内皮、平滑肌、癌细胞的粘附和增值，结合肝素、胰岛素、骨矿化结合素、血管内皮抑制因子、巨噬细胞集落刺激因子等生物活性物质，可用于医药、材料和生物医学领域。
近年来，人们主要从多种水产品或胎盘、肾等组织中提取出Ⅴ型胶原蛋白粗品。遗憾的是，Ⅴ型胶原蛋白在生物体内含量较低，属于次要胶原蛋白，提取过程较为复杂。同时，动物源性免疫反应也是导致胶原蛋白在应用中受限的重要原因。随着我国胶原蛋白产业的日益壮大，利用生物合成路径获得胶原蛋白日趋成熟，尤其是人源化胶原蛋白已经走在世界前列。2021年国家药监局对生物合成胶原蛋白做了命名分类，其中，重组人源化胶原蛋白是指由DNA重组技术制备的人胶原蛋白特定型别基因编码的全长或部分氨基酸序列片段，或是含人胶原蛋白功能片段的组合。
Ⅴ型胶原蛋白最早于人的胎盘和皮肤中被发现，含有三条不同强度、移动性的α链，会在组织中形成四种不同的亚型，即α1α1α2(Ⅴ)、α1α2α3(Ⅴ)、α3α3α3(Ⅴ)、Ⅴ型胶原蛋白α链和Ⅺ型胶原蛋白的α链的混合。三条不同的α链 组成三股绳状的前胶原蛋白分子，经细胞外的酶切处理后组装成细长的胶原纤维。细长的纤维在细胞周围的空间相互交联，交联后形成高强度的成熟Ⅴ型胶原蛋白纤维网；或者与Ⅺ型胶原蛋白等其他类型的胶原蛋白组成异三聚体，分布于角膜、皮肤、韧带、骨骼、肌腱、肌肉等部位。Ⅴ型胶原蛋白具有纤维状胶原蛋白的共同结构特征，在中央区域有一个超过1000个甘氨酸-Xaa-Yaa不间断的序列，其中Xaa-Yaa是任何氨基酸或者亚氨基酸残基。从结构上来说，人体天然的Ⅴ型胶原蛋白的结构非常复杂，所以才导致人源化胶原蛋白难以通过常规的手段表达和大量制备。
目前生产Ⅴ型胶原蛋白主要是通过胃蛋白酶来处理动物来源的组织，提取胶原蛋白衍生物。但是，这些方法提取的胶原蛋白已丧失了原本的生物学活性，无法发挥该胶原蛋白真正的功能。随着现代生物技术的发展，人们通过转基因技术目，已经可以在动物、植物、微生物表达体系中制备重组人源化胶原蛋白，解决了酶解法的缺点。但是目前，还未成功制备得到重组Ⅴ型人源化胶原蛋白。所以，亟需一种重组Ⅴ型人源化胶原蛋白的生物合成方法，使其作为人体结构性材料得到广泛应用。
发明内容
针对现有技术存在的技术问题，发明人首次设计了一种重组Ⅴ型人源化胶原蛋白的功能区筛选和蛋白合成工艺。发明人找到了Ⅴ型胶原蛋白的核心区的氨基酸序列(SEQ ID NO.1，gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv)，并且以该核心区为重复单元构建包含多个重复单元的多肽。发明人发现构建的多肽具备促进细胞黏附的活性。发明人还发现在核心区的N端和/或C端可以延长一个或多个氨基酸作为重复单元，构建的包含多个重复单元的多肽也具备促进细胞黏附的活性。进一步，在重复单元外，多肽还可以在重复单元外(例如重复单元构成的多肽的N端和/或C端)包含一定长度的肽段。该肽段可以是重复单元的从第1位起的连续氨基酸段。
在一方面，本申请提供了多肽，其包含(重复单元)n或(重复单元)n-C端区的结构，该重复单元包含SEQ ID NO.1的氨基酸序列，或者重复单元包含SEQ ID NO.1和SEQ ID NO.1的N端和/或C端的额外氨基酸残基，该额外氨基酸残基的数目是1-50。本申请的多肽是重组Ⅴ型人源化胶原蛋白。
在一个实施方案中，各重复单元是直接连接的并且重复单元的数目n是 4-20，并且C端区是重复单元的从第1位起的连续氨基酸段。
在一个实施方案中，额外氨基酸残基在SEQ ID NO.1的C端，并且是SEQ ID NO.7所示的氨基酸序列(galglk)或该氨基酸序列的连续氨基酸段。例如，额外氨基酸残基是SEQ ID NO.7所示的氨基酸序列的从第1位起的连续氨基酸段。
在一个实施方案中，重复单元包含SEQ ID NO.2、3和8-11中任一项的氨基酸序列。
在一个实施方案中，多肽包含选自SEQ ID NO.4-6的氨基酸序列。
在另一个方面，本申请提供了核酸，其包含本申请的多肽的核苷酸。
在一个实施方案中，核酸还包含编码纯化标签，例如His标签、GST标签、MBP标签、SUMO标签或NusA标签的核苷酸。
在一个实施方案中，核酸还包含编码前导序列的核苷酸。
在另一个方面，本申请提供了载体，其包含根据本申请的核酸。
在一个实施方案中，载体包含与核酸可操作连接的表达控制元件，如启动子、终止子和/或增强子。
在另一个方面，本申请提供了宿主细胞，其包含本申请的核酸或载体。在一个实施方案中，宿主细胞是真核细胞或原核细胞。在一个实施方案中，真核细胞是酵母细胞、动物细胞和/或昆虫细胞。在一个实施方案中，原核细胞是大肠杆菌细胞。
在另一个方面，本申请提供了生产本申请的多肽，其包括：
(1)在合适的培养条件下培养本申请的宿主细胞；
(2)收获包含多肽的宿主细胞和/或培养基；和
(3)纯化融合蛋白或三聚体融合蛋白。
在另一个方面，本申请提供了组合物，其包含本申请的多肽、核酸、载体和/或宿主细胞。
在一个实施方案中，组合物是生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种。
在另一个方面，本申请提供了本申请的多肽、核酸、载体、宿主细胞和 /或组合物在体外促进细胞黏附或者在制备促进细胞黏附的药物中的用途。
在另一个方面，本申请提供了本申请的多肽、核酸、载体、宿主细胞和/或组合物在高端医疗器械制备，如生物敷料、人体仿生材料、整形美容材料、类器官培养、心血管支架、涂层、组织注射填充、眼科材料、妇产科生物材料、神经修复再生、肝脏组织及血管修复再生、3D打印人造器官生物材料等；高端化妆品原料及高端药用辅料；食品添加剂中的应用。
本申请公开了一种重组Ⅴ型人源化胶原蛋白的功能区筛选和合成工艺的具体过程，可用作人体结构性材料制备。本申请属于合成生物技术领域。本申请公开了一种重组Ⅴ型人源化胶原蛋白的功能区筛选和合成工艺的具体过程，可用作人体结构性材料制备。本申请属于合成生物技术领域。本申请中制备重组Ⅴ型人源化胶原蛋白的生物合成方法，具体过程包括：(1)功能区筛选、菌种的构建；(2)生物发酵、诱导和表达；(3)人源化Ⅴ型胶原蛋白的纯化及任选的酶切。本申请制备的重组人源化胶原蛋白氨基酸序列源自人天然Ⅴ型胶原蛋白的功能区，包含该功能区及类似的功能区、对氨基酸序列分别进行突变和修饰后的蛋白。本申请制备得到的重组Ⅴ型人源化胶原蛋白具有高促进细胞黏附的活性，应用于人体不会产生免疫反应，且其制备方法新颖，可大规模获得重组Ⅴ型人源化胶原蛋白，在人体结构性材料制备方面得到广泛应用。其应用领域包含高端医疗器械制备，如生物敷料、人体仿生材料、整形美容材料、类器官培养、心血管支架、涂层、组织注射填充、眼科材料、妇产科生物材料、神经修复再生、肝脏组织及血管修复再生、3D打印人造器官生物材料等；高端化妆品原料及高端药用辅料；食品添加剂等。
本申请提供了以下内容：
1.针对目前的研究现状，本申请提供了一种生物合成重组Ⅴ型人源化胶原蛋白的方法，即人体结构性材料的制备方法，具体过程包括：(1)功能区筛选、菌种的构建；(2)大规模生物发酵培养和蛋白的诱导表达；(3)人源化Ⅴ型胶原蛋白的纯化和任选的酶切。
2.根据项1所述，功能区筛选、菌种的构建可如下进行：(1)大规模功能区筛选，得到目的基因功能区；(2)将得到的目的基因功能区插入pET-32a表达载体中得到重组表达质粒；(3)将重组表达质粒转入大肠杆菌感受态细胞BL21(DE3)中，筛选得到阳性大肠杆菌基因工程菌。
3.根据项1所述，大规模生物发酵可如下进行：将筛选得到的阳性大肠 杆菌基因工程菌加入抗生素储液的摇瓶中，并在220rpm、37℃恒温摇床中培养7h。
4.根据项1所述，蛋白的诱导表达可如下进行：(1)将培养后的摇瓶降温至16℃；(2)添加IPTG母液，进行诱导表达；(3)将诱导表达后的菌液装于离心瓶中，8000rpm、4℃离心10min后收集菌体。
5.根据项1所述，人源化Ⅴ型胶原蛋白的纯化和任选的酶切可如下进行：(1)在Ni亲和层析柱粗纯人源化Ⅴ型胶原蛋白；(2)按一定比例添加TEV酶酶切；(3)离子交换柱精纯人源化Ⅴ型胶原蛋白。
6.根据项2所述中，筛选出的功能区如下所示：(1)C5V3G1氨基酸序列：gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk；(2)C5V3G2氨基酸序列：gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal；(3)C5V3G3氨基酸序列：gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv。
7.本申请制备的重组Ⅴ型人源化胶原蛋白氨基酸序列源自人天然Ⅴ型胶原蛋白的功能区，包含该功能区及类似的功能区、对氨基酸序列分别进行突变和修饰后的蛋白。
8.本申请制备得到的重组Ⅴ型人源化胶原蛋白，其应用领域包含高端医疗器械制备，如生物敷料、人体仿生材料、整形美容材料、类器官培养、心血管支架、涂层、心肌修复、组织注射填充、眼科材料、妇产科生物材料、 神经修复再生、肝脏组织及血管修复再生、3D打印人造器官生物材料、肿瘤预防、骨修复材料、皮肤修复材料、肾组织修复、胰腺修复等；高端化妆品原料及高端药用辅料；食品添加剂等。
相对于现有技术，本申请的优点包括：
1.本申请提供了重组Ⅴ型人源化胶原蛋白的核心功能区及氨基酸序列；
2.本申请首次成功生物合成了重组Ⅴ型人源化胶原蛋白；
3.本申请制备得到的重组Ⅴ型人源化胶原蛋白具有良好的细胞黏附效果，应用于人体不会产生免疫反应；
4.其制备方法简单，可大规模获得重组Ⅴ型人源化胶原蛋白。
附图说明
图1：重组Ⅴ型人源化胶原蛋白C5V3G1和C5V3G2粗纯后的电泳检测图。显示了各纯化步骤后的电泳图。
图2：重组Ⅴ型人源化胶原蛋白C5V3G3粗纯后的电泳检测图和C5V3G1、C5V3G2、C5V3G3酶切后的电泳检测图。显示了各纯化步骤后的电泳图。
图3：重组Ⅴ型人源化胶原蛋白C5V3G1、C5V3G2、C5V3G3精纯后的电泳检测图。
图4：重组Ⅴ型人源化胶原蛋白C5V3G1的细胞黏附活性。
图5：重组Ⅴ型人源化胶原蛋白C5V3G2的细胞黏附活性。
图6：重组Ⅴ型人源化胶原蛋白C5V3G3的细胞黏附活性。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚，下面将结合本申请的实施例，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
如本文中所用，多肽是指通过肽键连接的多个氨基酸残基。在本文中，多肽包含多个重复单元，该重复单元来自人Ⅴ型胶原蛋白(GenBank:KAI4009058.1)。多肽可以包含含有(重复单元)n或(重复单元)n-C端区的结构，该重复单元包含SEQ ID NO.1的氨基酸序列，或者重复单元包含SEQ ID NO. 1和SEQ ID NO.1的N端和/或C端的额外氨基酸残基，该额外氨基酸残基的数目是1-50。例如，该额外氨基酸残基的数目是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48或49。该额外氨基酸残基可以来自人Ⅴ型胶原蛋白。例如，该额外氨基酸残基可以是重复单元在天然人Ⅴ型胶原蛋白中原位直接相邻的连续氨基酸残基。该额外氨基酸残基可以是SEQ ID NO.7所示的氨基酸序列或该氨基酸序列的连续氨基酸段。优选地，该额外氨基酸残基可以是SEQ ID NO.7所示的氨基酸序列的从第1位起的连续氨基酸段。在本文中，该额外氨基酸残基可以位于C端。
在本文中，各重复单元可以是直接连接的或者可以间隔一个或多个氨基酸残基。重复单元的数目n可以是4-20，例如5、6、7、8、9、10、11、12、13、14、15、16、17、18或19。多肽可以包含C端区。该C端区可以是重复单元的从第1位起的连续氨基酸段。重复单元可以包含SEQ ID NO.2或3中任一项的氨基酸序列。
如本文中所用，在结合氨基酸使用的表述“段”是指小于指定序列的部分序列。例如，SEQ ID NO.7中的氨基酸段是指小于SEQ ID NO.7的长度的任何长度的部分序列。“连续氨基酸段”是指指定序列中直接相邻的氨基酸构成的氨基酸段。
如本文中所用，就多肽或特定氨基酸序列而言，“C端”和“N端”是指相对于该多肽或特定氨基酸序列的位置，具体指位于多肽或特定氨基酸序列的羧基端或氨基端方向。
如本文中所用，当提及多肽或特定氨基酸序列时，位置是相对于多肽或特定氨基酸序列的C端描述的。例如，当提到“SEQ ID NO.7的氨基酸序列(galglk)的第1位起”时，第1位氨基酸是指G。
在本文中，本申请的重复单元可以包含或者可以是以下序列：
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv(SEQ ID NO.1)；
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal(SEQ ID NO.2)；
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk(SEQ ID NO.3)；
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvg(SEQ ID NO.8)；
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvga(SEQ ID NO.9)；
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalg(SEQ ID NO.10)；
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgl(SEQ ID NO.11)。
如本文中所用，“核酸”是指通过核苷酸间连接的多个核苷酸。核苷酸间连接可以是例如磷酸二酯键。本文中的核酸可以包含编码本申请的多肽的多核苷酸。为了便于多肽后续处理，本申请的核酸还可以包含编码纯化标签，例如His标签、GST标签、MBP标签、SUMO标签或NusA标签的核苷酸，以及当需要时编码前导序列的核苷酸。
如本文中所用，术语“载体”是可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白质获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。载体可以含有多种控制表达的元件，包括但不限于启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体可以包含本申请的核酸以便于导入细胞进行表达。载体可以包含与所述核酸可操作连接的表达控制元件，如启动子、终止子和/或增强子。
如本文中所用，术语“宿主细胞”是已经通过分子生物学技术将核酸分子引入的细胞。这些技术包括转染病毒载体，用质粒载体转化，以及通过电穿孔、脂转染、和粒子枪加速引入裸DNA。宿主细胞可以是真核细胞或原核细胞。例如，真核细胞是酵母细胞、动物细胞和/或昆虫细胞。原核细胞可以是大肠杆菌细胞。
在本文中，本申请的多肽的各个部分，例如重复单元或C端区可以存在一定的突变。例如，这些部分中的一个或多个的氨基酸序列可以存在氨基酸残基的取代、缺失、添加或插入。也就是说，本申请可以使用各个部分的变体，例如重复单元变体或C端区变体，只要变体保留促进细胞黏附的活性。具体而言，变体可以与指定的序列具备一定的百分比同一性，例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。指定的序列可以是本申请的任何序列，例如SEQ ID NO.1-6，但是优选的是这些变体保留本申请鉴定的核心序列。
本申请的多肽可以通过任何合适的方式进行制备，例如可以通过合成制备。优选地，本申请的多肽可以通过重组方式制备。作为制备方法，该方法可以包括以下任何一个或多个步骤：(1)在合适的培养条件下培养根据权利要求8所述的宿主细胞；(2)收获包含多肽的宿主细胞和/或培养基；和(3)纯化融合蛋白或三聚体融合蛋白。更具体而言，可以将本申请的多肽的编码序列插入到合适的表达载体，诸如PET-28a中，以得到重组表达载体。可以将重组表达质粒转入大肠杆菌感受态细胞BL21(DE3)中，筛选得到阳性大肠杆菌基因工程菌。然后，可以将筛选得到的阳性大肠杆菌基因工程菌进行大规模生物发酵(例如在220rpm、37℃恒温摇床中培养7h)。在培养到合适的细胞密度时，可以诱导细胞表达蛋白质。例如，在大肠杆菌基因工程菌的情况下，(1)将培养后的摇瓶降温至16℃；(2)添加IPTG母液，进行诱导表达；(3)将诱导表达后的菌液装于离心瓶中，8000rpm、4℃离心10min后收集菌体。可以对菌体进行裂解(例如通过高压均质)以得到菌体裂解物，然后离心得到上清液。可以对上清液进行纯化得到纯化的多肽。例如，纯化过程可以包括以下一个或多个步骤：(1)在Ni亲和层析柱粗纯多肽；(2)按一定比例添加TEV酶酶切；和(3)离子交换柱精纯多肽。
本申请的多肽、核酸、载体和/或宿主细胞可以制备成组合物或试剂盒。组合物或试剂盒可以包含生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种。组合物可以是生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种。该组合物或试剂盒可以用于在体外促进细胞黏附或者在体内促进细胞黏附。
实施例
提供以下实施例来阐述本申请。本领域技术人员应当理解实施例仅仅是例示性的，而非限制性的。本申请仅仅由所附权利要求书的范围限定。
实施例1：重组Ⅴ型人源化胶原蛋白构建及表达
1.进行大规模功能区筛选，得到以下不同的重组Ⅴ型人源化胶原蛋白的目的基因片段：(1)C5V3G1氨基酸序列：
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk(C5V3G1氨基酸序列：SEQ ID NO.6；重复序列SEQ ID NO.3,gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk)；(2)C5V3G2氨基酸序列：
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal(C5V3G2氨基酸序列SEQ ID NO:5，重复单元SEQ ID NO.2,gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal)；(3)C5V3G3氨基酸序列：gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv
gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv(C5V3G3氨基酸序列：SEQ ID NO:4， 重复单元为SEQ ID NO.1：gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv)。
将合成的基因片段(C5V3G1碱基序列：GGGAAAGAAGGAACGAAAGGTGACCCTGGCCCAGCGGGTCTGCCGGGCAAGGACGGTCCACCGGGTCTGCGCGGTTTCCCGGGCGACCGCGGCCTGCCGGGTCCGGTTGGCGCGTTGGGTCTTAAGGGCAAAGAAGGCACCAAAGGAGATCCGGGTCCGGCTGGTTTACCGGGCAAGGATGGTCCGCCGGGTCTGCGTGGTTTTCCGGGTGACCGTGGTCTGCCGGGTCCGGTCGGAGCCCTGGGTTTGAAGGGCAAAGAGGGCACCAAAGGCGACCCAGGACCGGCAGGCCTCCCGGGTAAGGATGGCCCTCCGGGGCTGCGTGGTTTTCCGGGTGATCGCGGCTTGCCGGGACCGGTTGGTGCGCTGGGTCTTAAGGGTAAAGAAGGCACCAAAGGTGACCCGGGCCCGGCGGGTCTGCCGGGCAAAGATGGTCCGCCAGGCCTGAGAGGCTTCCCGGGTGACCGCGGCTTGCCGGGTCCGGTTGGTGCGCTGGGCCTTAAGGGTAAAGAGGGCACCAAAGGCGATCCGGGTCCGGCTGGCCTGCCGGGGAAGGACGGCCCACCGGGCCTGCGTGGTTTTCCGGGCGACCGTGGTTTGCCGGGCCCGGTGGGCGCTCTGGGTCTGAAAGGGAAAGAGGGTACGAAGGGTGATCCGGGTCCGGCAGGCTTGCCGGGCAAGGACGGCCCGCCTGGCCTGCGTGGTTTCCCGGGTGACCGTGGTTTACCGGGTCCCGTGGGCGCGCTGGGTCTGAAAGGTAAGGAAGGCACCAAGGGTGATCCGGGCCCGGCCGGTCTGCCGGGCAAGGACGGTCCGCCCGGATTACGTGGCTTCCCGGGTGATCGTGGTCTGCCGGGTCCTGTAGGTGCGCTGGGTCTCAAGGGCAAAGAGGGTACTAAGGGTGATCCGGGCCCGGCGGGTCTGCCAGGCAAAGACGGCCCACCGGGCTTGCGCGGTTTTCCGGGCGATCGTGGTTTGCCGGGTCCTGTGGGCGCACTGGGCTTGAAGTAA，SEQ ID NO:12；C5V3G2碱基序列：GGGAAAGAAGGAACCAAGGGTGACCCGGGGCCAGCCGGTTTGCCGGGTAAGGATGGTCCGCCTGGCTTGCGTGGTTTTCCGGGGGACCGTGGTTTGCCGGGTCCGGTTGGTGCGCTGGGTAAAGAAGGCACGAAGGGCGACCCGGGCCCAGCCGGTTTACCGGGGAAAGATGGTCCTCCGGGCCTGCGCGGTTTTCCGGGAGACCGTGGTCTTCCGGGCCCGGTGGGTGCGCTGGGCAAGGAGGGCACCAAAGGTGACCCTGGCCCTGCGGGTCTGCCGGGTAAGGACGGTCCACCGGGTCTGCGTGGCTTTCCGGGCGACCGTGGCTTGCCCGGTCCGGTTGGCGCGCTGGGTAAAGAGGGTACGAAGGGTGACCCGGGTCCTGCGGGCCTGCCGGGCAAGGACGGCCCGCCGGGACTGCGCGGATTCCCGGGCGATCGCGGCCTCCCGGGTCCGGTCGGTGCGCTGGGCAAAGAAGGTACTAAAGGTGATCCGGGCCCGGCGGGCCTGCCGGGCAAGGATGGCCCGCCGGGTTTGCGCGGTTTCCCGGGCGACCGTGGTTTGCCGGGCCCAGTGGGCGCACTGGGTAAGGAGGGCACCAAAGGCGATCCGGGACCAGCTGGTCTGCCGGGCAAGGACGGCCCACCGGGTCTCAGAGGCTTCCCGGGGGACCGCGGTCTGCCGGGCCCGGTAGGCGCACTGGGTAAAGAAGGCACCAAAGGTGACCCGGGTCCGGCTGGTCTGCCGGGTAAGGATGGCCCGCCGGGCCTGCGTGGCTTCCCGGGTGATCGTGGCTTGCCTGGCCCAGTGGGTGCACTGGGCAAAGAGGGCACCAAGGGTGATCCGGGTCCGGCGGGTCTGCCGGGTAAAGATGGTCCACCGGGTTTACGTGGTTTTCCGGGCGATCGTGGTCTCCCGGGCCCGGTTGGCGCTCTGTAA，SEQ ID NO:13；C5V3G3碱基序列：GGGAAAGAAGGAACGAAAGGCGATCCGGGTCCGGCGGGCCTGCCGGGTAAGGACGGCCCGCCGGGATTACGCGGTTTCCCGGGTGACCGCGGTTTGCCGGGTCCGGTTGGCAAAGAAGGTACTAAGGGTGATCCGGGCCCAGCCGGTCTGCCGGGCAAGGATGGTCCGCCAGGCCTGAGAGGCTTCCCGGGTGACCGCGGGCTGCCGGGCCCGGTTGGTAAGGAGGGCACCAAAGGCGATCCGGGTCCGGCGGGTTTGCCGGGCAAGGATGGCCCGCCAGGCCTGCGTGGCTTTCCGGGTGATCGTGGTCTGCCGGGCCCCGTGGGCAAAGAGGGCACCAAAGGTGATCCGGGTCCAGCGGGTCTGCCAGGTAAGGACGGTCCGCCTGGCCTGCGTGGTTTCCCGGGTGATCGTGGTCTGCCGGGTCCGGTCGGTAAAGAAGGCACCAAGGGCGACCCGGGCCCGGCAGGGCTGCCGGGTAAGGATGGCCCACCGGGTCTGCGTGGTTTTCCGGGCGACCGTGGTCTTCCGGGTCCGGTCGGTAAAGAGGGCACCAAAGGTGACCCGGGTCCCGCGGGTCTGCCGGGTAAGGACGGCCCTCCGGGCCTGCGCGGCTTCCCGGGCGATCGCGGTCTGCCGGGTCCGGTTGGTAAAGAAGGTA CGAAGGGCGATCCTGGTCCAGCAGGCTTGCCGGGCAAAGATGGTCCGCCCGGCCTTCGCGGATTCCCGGGCGACCGTGGCCTGCCGGGTCCGGTGGGTAAAGAAGGCACCAAGGGCGACCCGGGTCCTGCTGGCTTGCCGGGCAAGGACGGCCCACCGGGTTTACGCGGTTTTCCGGGCGATCGTGGTTTGCCGGGTCCGGTGGGCAAAGAGGGAACCAAAGGTGACCCTGGCCCAGCTGGCTTGCCGGGCAAAGATGGTCCGCCGGGTCTGCGTGGCTTTCCGGGTGACCGTGGTTTGCCGGGCCCGGTTGGTAAGGAGGGCACCAAAGGCGACCCGGGTCCGGCGGGTCTGCCGGGTAAGGACGGTCCACCGGGCTTACGTGGTTTCCCGGGGGACCGTGGCCTGCCGGGCCCCGTGTAA，SEQ ID NO:14)插入pET-32a表达载体的Kpn I和Xho I酶切位点之间，得到重组表达质粒。
2.将构建成功的表达质粒转化大肠杆菌感受态细胞BL21(DE3)。具体过程为：(1)在超低温冰箱中取出大肠杆菌感受态细胞BL21(DE3)置于冰上，待半融时取2μl待转化的质粒加入大肠杆菌感受态细胞BL21(DE3)中，稍微混匀2-3次。(2)将混合物置于冰上冰浴30min，然后于42℃水浴热激45-90s，取出后置于冰上冰浴2min。(3)转移至生物安全柜中，并加入700μl液体LB培养基，然后于37℃、220rpm条件下培养60min。(4)取200μl的菌液均匀涂布在含有氨苄西林钠的LB平板上。(5)将平板在37℃的培养箱中培养15-17h，待其长出大小均匀的菌落。
3.从转化好的LB平板中挑取5-6个单菌落于含有抗生素储液的摇瓶中，在220rpm，37℃恒温摇床中7h。再将培养后的摇瓶降温至16℃，添加IPTG诱导表达一段时间后，将菌液分装于离心瓶中，于8000rpm、4℃离心10min，收集菌体，并记录菌体重量，取样进行电泳检测(见图1和图2中的“菌体”泳道)。
4.将收集的菌体用平衡工作液重悬，将菌液降温至≤15℃，进行均质，高压均质两次，完成后收集菌液。将均质后的菌液分装至离心瓶中，于17000rpm、4℃离心30min，收集上清液，取上清液和沉淀进行电泳检测(见图1和图2，“均二”为高压均质两次后菌液的电泳图，“上清”和“沉淀”分别为上清液和沉淀的电泳图)。
5.将重组人源化Ⅴ型胶原蛋白的进行纯化和酶切，具体过程是：(1)粗纯：a.水洗柱材。b.平衡柱材。c.上样：将离心后的上清液加入柱材中，直至液体流完后，取流穿进行电泳检验(见图1和图2，“流穿”泳道)。d.清洗杂蛋白：添加洗杂液25mL(200mM氯化钠，25mM Tris，20mM咪唑)至液体流完，并取洗杂流穿进行电泳检验(见图1和图2，“洗杂”泳道)。e.收集目的蛋白：添加20mL洗脱液，并收集流穿液，获得含His标签的目的蛋白，采用紫外可见分光光度法检测蛋白浓度，按以下公式(C(mg/ml) ＝A280×稀释倍数×消光系数)计算蛋白浓度，并进行电泳检测(见图1和图2，“洗脱”泳道)。f.用1M咪唑工作液清洗柱材。g.纯化水清洗柱材。(2)酶切：按蛋白总量与TEV酶总量比为20：1，添加TEV酶，16℃酶切2h，取样进行电泳检测。将酶切后的蛋白液放入透析袋，于4℃透析2h，再转移至新的透析液中4℃过夜透析(换液后的电泳图见图3)。(3)精纯：a.平衡柱材：使用A液(20mM Tris，20mM氯化钠)将柱材平衡，流速为10ml/min。b.上样：流速为5ml/min，上样并收集流穿(标记FL1，见图3)，获得重组Ⅴ型人源化胶原蛋白C5V3G1、C5V3G2和C5V3G3，并进行电泳检测，如图3。c.洗脱：设置100％B液(20mM Tris，1M氯化钠，pH8.0)洗脱3个CV，出峰收集并进行电泳检测(B液洗脱液见图3的“B液洗”泳道)。d.清洗柱材。e.将蛋白储存在4℃环境中。图3显示了重组Ⅴ型人源化胶原蛋白C5V3G1、C5V3G2和C5V3G3的表观分子量为分别为32kDa、29kDa和34kDa，与预测的分子量相符。
实施例2：重组Ⅴ型人源化胶原蛋白的生物活性检测
胶原蛋白的活性检测方法可以参考文献Juming Yao,Satoshi Yanagisawa,Tetsuo Asakura,Design,Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens,J Biochem.136,643-649(2004)。具体实施方法如下：
(1)利用紫外吸收法检测待测蛋白样品的浓度，包括牛Ⅰ型胶原蛋白(中国食品药品检定研究院，编号：380002；0.5mg/mL，阳性孔)、本申请提供的重组Ⅴ型人源化胶原蛋白C5V3G1、C5V3G2、C5V3G3。
具体为分别测定样品在215nm和225nm下的紫外光吸收，利用经验公式C(μg/mL)＝144×(A215-A225)计算蛋白质浓度，注意需在A215<1.5的情况下检测。该方法的原理是：测定肽键在远紫外光下的特征吸收，不受生色团含量的影响，干扰物质少，操作简便，适合检测考马斯亮蓝不显色的人胶原蛋白及其类似物。(参考文献为Walker JM.The Protein Protocols Handbook,second edition.HumanaPress.43-45.)。检测完蛋白浓度后，用PBS将所有待测蛋白浓度调整到0.25mg/mL、0.5mg/mL、或1mg/ml。
(2)向96孔板中加入100μL各种蛋白溶液和空白PBS溶液对照(见图4-图6中的D-PBS组)，室温静置60min。
(3)每孔中加入105个培养状态良好的3T3细胞，37℃孵育60min。
(4)每孔用PBS清洗4次。
(5)用LDH检测试剂盒(Roche，04744926001)检测OD492nm的吸光度。根据空白对照的数值，可以计算出细胞的贴壁率。计算公式如下：细胞的贴壁率(相对细胞粘附活性)即可以反应胶原蛋白的活性。蛋白的活性越高，越能在短时间给细胞提供优质的外环境，帮助细胞贴壁。
结果如图4、5、6所示，从对比中可知，相比于牛Ⅰ型胶原蛋白(B ColI组，0.5mg/ml)，本申请的重组Ⅴ型人源化胶原蛋白具有更加优秀的生物黏附活性。图4描述了C5V3G1相对于牛Ⅰ型胶原蛋白的相对细胞黏附活性，表明C5V3G1具有细胞黏附活性，并且在0.25、0.5和1mg/ml的浓度比牛Ⅰ型胶原蛋白具有更高的细胞黏附活性。图5显示了C5V3G2相对于牛Ⅰ型胶原蛋白的相对细胞黏附活性，表明0.25mg/ml以上的浓度C5V3G2具有更好的细胞黏附活性。图6显示了C5V3G3相对于牛Ⅰ型胶原蛋白的相对细胞黏附活性，表明C5V3G3具有细胞黏附活性，并且在0.5mg/ml的浓度比牛Ⅰ型胶原蛋白具有更高的细胞黏附活性。
实施例3：重组Ⅴ型人源化胶原蛋白的质谱检测
实验方法
蛋白样品经DTT还原和碘代乙酰胺烷基化处理后，加入胰蛋白酶酶解过夜。酶解后得到的肽段再经C18ZipTip脱盐后，与基质α-cyano-4-hydroxycinnamic acid(CHCA)混合点板。最后用基质辅助激光解析电离-飞行时间质谱仪MALDI-TOF/TOF UlraflextremeTM，Brucker，Germany进行分析(肽指纹图谱的技术可以参考Protein J.2016；35:212-7)。 数据检索是通过从本地masco网站上MS/MS Ion Search页面处理的。蛋白质鉴定结果是根据酶解后所产生的肽段的一级质谱得到的。检测参数：Trypsin酶解，设两个漏切位点。设定半胱氨酸的烷基化为固定修饰。甲硫氨酸的氧化为可变修饰。鉴定所用的数据库为NCBprot。
表1：C5V3G1质谱检出分子量及对应多肽
检出多肽片段的覆盖率为100％，与理论序列一致，检测结果非常可信。
表2：C5V3G2质谱检出分子量及对应多肽
检出多肽片段与理论序列相比，覆盖率为83％，检测结果非常可信。。
表3：C5V3G3质谱检出分子量及对应多肽
检出多肽片段与理论序列相比，覆盖率为96.11％，检测结果非常可信。
上述实施例为本申请较佳的实施方式，但本申请的实施方式并不受上述实施例的限制，其他的任何未背离本申请的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本申请的保护 范围之内。

Claims (13)

  1. 多肽，其包含(重复单元)n或(重复单元)n-C端区的结构，该重复单元包含SEQ ID NO.1的氨基酸序列，或者重复单元包含SEQ ID NO.1和SEQ ID NO.1的N端和/或C端的额外氨基酸残基，该额外氨基酸残基的数目是1-50；
    各重复单元是直接连接的并且重复单元的数目n是4-20，并且C端区是重复单元的从第1位起的连续氨基酸段；优选地所述多肽是重组Ⅴ型人源化胶原蛋白。
  2. 根据权利要求1所述的多肽，其中额外氨基酸残基在SEQ ID NO.1的C端，并且是SEQ ID NO.7所示的氨基酸序列或该氨基酸序列的连续氨基酸段。
  3. 根据权利要求2所述的多肽，其中额外氨基酸残基是SEQ ID NO.7的氨基酸序列的第1位起的连续氨基酸段。
  4. 根据权利要求1-3中任一项所述的多肽，其中重复单元包含SEQ ID NO.2、3和8-11中任一项的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的多肽，其包含选自SEQ ID NO.4-6的氨基酸序列。
  6. 核酸，其包含编码权利要求1-5中任一项的多肽的核苷酸，任选地，核酸还包含编码纯化标签，例如His标签、GST标签、MBP标签、SUMO标签或NusA标签的核苷酸；任选地，核酸还包含编码前导序列的核苷酸。
  7. 载体，其包含根据权利要求6所述的核酸，任选地包含与所述核酸可操作连接的表达控制元件，如启动子、终止子和/或增强子。
  8. 宿主细胞，其包含根据权利要求6所述的核酸或根据权利要求7所述的载体；其中优选地，宿主细胞是真核细胞或原核细胞；其中优选地，真核细胞是酵母细胞、动物细胞和/或昆虫细胞，和/或原核细胞是大肠杆菌细胞，例如大肠杆菌BL21。
  9. 生产根据权利要求1-5中任一项所述的多肽，其包括：
    (1)在合适的培养条件下培养根据权利要求8所述的宿主细胞；
    (2)收获包含多肽的宿主细胞和/或培养基；和
    (3)纯化融合蛋白，例如(1)在Ni亲和层析柱粗纯多肽；(2)按一定比例添加TEV酶酶切；(3)离子交换柱精纯多肽。
  10. 组合物，其包含根据权利要求1-5中任一项所述的多肽、根据权利要 求6所述的核酸、根据权利要求7所述的载体和/或根据权利要求8所述的宿主细胞。
  11. 根据权利要求10所述的组合物，其是生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种。
  12. 根据权利要求1-5中任一项所述的多肽、根据权利要求6所述的核酸、根据权利要求7所述的载体、根据权利要求8所述的宿主细胞和/或根据权利要求10所述的组合物在体外促进细胞黏附或者在制备促进细胞黏附的产品或试剂盒中的用途。
  13. 根据权利要求1-5中任一项所述的多肽、根据权利要求6所述的核酸、根据权利要求7所述的载体、根据权利要求8所述的宿主细胞和/或根据权利要求10所述的组合物在高端医疗器械制备，如生物敷料、人体仿生材料、整形美容材料、类器官培养、心血管支架、涂层、组织注射填充、眼科材料、妇产科生物材料、神经修复再生、肝脏组织及血管修复再生、3D打印人造器官生物材料等；高端化妆品原料及高端药用辅料；食品添加剂中的应用。
PCT/CN2023/087212 2022-07-19 2023-04-10 一种生物合成人体结构性材料的制备方法 WO2024016740A1 (zh)

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