WO2024234609A1 - 胶原蛋白及其用途 - Google Patents

胶原蛋白及其用途 Download PDF

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Publication number
WO2024234609A1
WO2024234609A1 PCT/CN2023/134277 CN2023134277W WO2024234609A1 WO 2024234609 A1 WO2024234609 A1 WO 2024234609A1 CN 2023134277 W CN2023134277 W CN 2023134277W WO 2024234609 A1 WO2024234609 A1 WO 2024234609A1
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Prior art keywords
collagen
amino acid
materials
acid sequence
composition
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English (en)
French (fr)
Inventor
杨霞
兰小宾
王玲玲
张永健
刘欣
刘增耀
杨文飞
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Shanxi Jinbo Bio Pharmaceutical Co Ltd
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Shanxi Jinbo Bio Pharmaceutical Co Ltd
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Priority to JP2024570537A priority Critical patent/JP7746608B2/ja
Priority to EP23936793.1A priority patent/EP4512825A4/en
Priority to KR1020247040329A priority patent/KR102832050B1/ko
Priority to US18/933,318 priority patent/US12479906B2/en
Publication of WO2024234609A1 publication Critical patent/WO2024234609A1/zh
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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
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0033—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
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/044—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • 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

  • the present invention belongs to the field of synthetic biotechnology, and specifically relates to human body structural materials and their biosynthetic preparation methods.
  • 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. It has been found that there are at least 28 collagen subtypes in the human body, which are located in different tissues and organs.
  • Type II collagen is mainly found in cartilage tissue, vitreous body and cornea. It is a high molecular weight protein. Its filamentous collagen fibers interweave with elastin and polysaccharide proteins to form a network structure, also known as composite bone collagen. Type II collagen is a necessary component for cartilage and bone formation, bone growth and mature cartilage maintenance, so non-denatured collagen can be used as a structural and functional component of cartilage.
  • Type II collagen as the main component of the articular cartilage matrix, protects the cartilage from wear and tear together with lubricating components such as hyaluronic acid and proteoglycans. With age, the synthesis rate of collagen gradually decreases, the loss of collagen intensifies, the articular cartilage degenerates, and the friction between bones intensifies, which can cause inflammation in the joints.
  • the common treatment for osteoarthritis is to eat collagen, but its protein utilization rate is low, resulting in slow cartilage regeneration, which is not conducive to the patient's recovery and affects the progress and efficiency of treatment. Because the current treatment methods are not ideal, patients often have to choose artificial joint replacement surgery, which is not only expensive, but also may have catastrophic complications such as postoperative thrombosis, post-replacement infection and amputation.
  • the collagen material currently used as cartilage is mainly derived from animal cartilage extraction.
  • the immunogenicity of animal-derived collagen cannot be eliminated, and the impurity removal and extraction process is still relatively complicated. It is difficult to ensure the triple helix structure of collagen, and it cannot be used for large-scale production.
  • recombinant humanized collagen refers to the full-length or partial amino acid sequence functional region encoded by the specific type gene of human collagen prepared by DNA recombinant technology, or a combination of functional regions containing human collagen functions.
  • the traditional method of producing collagen is to use acid, alkali, and enzymatic methods to treat animal tissues and extract collagen derivatives.
  • the collagen extracted by these methods has lost its original biological activity and cannot be used in the biomedical field to play a real role.
  • some extraction methods have emerged at home and abroad to remove impurities and extract animal cartilage by enzymatic hydrolysis to obtain non-denatured type II collagen.
  • the collagen prepared by this method can be used for cartilage repair, the time for impurity removal and extraction is long, the product purity is low, and the product stability is poor, which is not conducive to large-scale production.
  • a company proposed to use Pichia yeast as a host strain to prepare type II collagen for cartilage repair but this collagen does not belong to humanized collagen, so it has a certain immunogenic reaction.
  • Human structural materials are mainly structural proteins including collagen. This type of protein has a complex structure and precise function. It is of great significance to the repair and regeneration of human tissues, but it is not easy to obtain using conventional production methods.
  • the present invention uses synthetic biology and structural biology technology to develop a macromolecular functional protein with a triple helix structure of human type II collagen and the function of human collagen.
  • the inventors conducted large-scale functional region screening. First, sequence screening.
  • the Gly-X-Y repeating gene sequence in the helical region of natural type II collagen contains a large number of charged amino acids.
  • the present invention includes the core functional region screening, synthesis process and Application scenarios: This patented design is the first to invent a functional region screening and protein synthesis process for recombinant type II humanized collagen that is directly injected into the human body and can be used for cartilage repair.
  • the present invention is based in part on the following unexpected findings by the inventors: Compared with other recombinant humanized type II collagens, recombinant humanized type II collagen HC2B-A10 is expressed in higher amounts when recombinantly expressed, is easier to separate and purify, and has superior cell adhesion effects compared with bovine type I collagen.
  • the present invention provides a collagen or polypeptide comprising a plurality of repeating units, the repeating unit comprising the amino acid sequence shown in SEQ ID NO.15 (gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak) or a variant thereof having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, and each repeating unit is directly connected or separated by one or more amino acid residues.
  • the repeating unit may be an amino acid sequence obtained by mutation (substitution, insertion, deletion or addition) of one or more amino acid residues in the amino acid sequence of SEQ ID NO.15.
  • the number of repeating units is 1-20. In one embodiment, the number of repeating units is 6-10. In one embodiment, the number of repeating units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the collagen or polypeptide comprises the amino acid sequence shown in SEQ ID NO.10.
  • the collagen or polypeptide comprises the amino acid sequence shown in SEQ ID NO.10 or the amino acid sequence of SEQ ID NO.3 or 10 obtained by mutation (substitution, insertion, deletion or addition) of one or more amino acid residues, or a variant having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO.10.
  • the obtained collagen or polypeptide retains the function of the present invention, such as the ability of cell adhesion and cartilage repair.
  • the present invention provides polynucleotides encoding the collagen or polypeptide described herein.
  • the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.29.
  • the invention provides a nucleic acid comprising a polynucleotide as described herein.
  • 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.
  • the nucleic acid further comprises nucleotides encoding a leader sequence.
  • the invention provides a vector comprising a polynucleotide described herein or a nucleic acid described herein.
  • the vector is an expression vector.
  • the vector comprises expression control elements, such as a promoter, terminator and/or enhancer, operably linked to the polynucleotide or nucleic acid.
  • the invention provides a host cell comprising a polynucleotide as described herein, a nucleic acid as described herein, or a vector as described herein.
  • the host cell is a bacterium, a fungus, or an animal cell.
  • the bacterium is Escherichia coli.
  • the fungus is a yeast, such as Saccharomyces cerevisiae.
  • the present invention provides a method for producing the collagen or polypeptide described herein, comprising:
  • the present invention provides a composition comprising collagen or polypeptide as described herein.
  • the composition is a kit.
  • the composition is a biological dressing, a human bionic material, a plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a 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 pharmaceutical excipient, and a food additive.
  • the composition is an injectable composition or an oral composition.
  • the composition is a composition for cartilage repair. In one embodiment, the composition is an injectable composition for cartilage repair.
  • the present invention provides a method for increasing cell adhesion, comprising contacting a cell with a collagen or polypeptide described herein, a polynucleotide described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, and/or a composition described herein.
  • the present invention provides the use of the collagen or polypeptide described herein, the polynucleotide described herein, the nucleic acid described herein, the vector described herein, the host cell described herein, and/or the composition described herein in the preparation of a kit for increasing cell adhesion or cartilage repair.
  • the present invention provides the use of the collagen or polypeptide described herein, the polynucleotide described herein, the nucleic acid described herein, the vector described herein, the host cell described herein, and/or the composition described herein in 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 regeneration materials, liver tissue materials and vascular repair regeneration materials, 3D printing Use in one or more of artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.
  • the present invention provides the core functional region and amino acid sequence of recombinant type II humanized collagen.
  • the present invention successfully synthesized for the first time a recombinant humanized type II collagen that can be injected into the human body for use in cartilage repair.
  • the amino acid composition of the recombinant humanized type II collagen produced is 100% identical to the corresponding part of the amino acid sequence of natural collagen, and will not cause immune rejection and allergic reactions when applied to the human body.
  • the preparation method in this article is simple and can be used to mass-produce high-yield recombinant type II humanized collagen.
  • recombinant type II humanized collagen HC2B-A10 achieved the best effect.
  • the recombinant humanized type II collagen of the present invention has more excellent bioadhesion activity, that is, the recombinant humanized type II collagen HC2B-A10 is greater than bovine type I collagen.
  • Figure 1 Crude purity of recombinant humanized type II collagen HC2B-a6 and HC2B-b5;
  • Figure 2 Crude purity of recombinant humanized type II collagen HC2B-b1, HC2B-a3 and HC2B-b3;
  • Figure 3 Purification of recombinant humanized type II collagen B3Q, HC2B-A8 and HC2B-A7;
  • Figure 4 Purification of recombinant type II humanized collagen HC2B-A10 and crude purity of HC2B-B9;
  • Figure 5 Purification of recombinant type II humanized collagen HC2B-A9;
  • FIG6 Crude purity of recombinant humanized type II collagen HC2B-B8, HC2B-B7 and HC2B-B10;
  • FIG7 Purification of recombinant humanized type II collagen HC2B-B7 and HC2B-B9;
  • Figure 8 Activity detection of recombinant type II humanized collagen HC2B-A10;
  • Figure 9 Map of pET-28a-Trx-His expression vector.
  • Recombinant collagen is a new type of biomaterial that uses cutting-edge structural biology, genetic engineering and other technologies to screen and prepare the genetic coding of the functional region of a specific type of human collagen, which has the same or similar amino acid sequence as human collagen.
  • polypeptide refers to a plurality of amino acid residues connected by peptide bonds.
  • collagen, recombinant collagen, recombinant type II humanized collagen or polypeptide can be used interchangeably.
  • collagen or polypeptide may comprise one or more repeating units, the repeating unit comprising the amino acid sequence shown in SEQ ID NO.15 or the amino acid sequence after mutation (substitution, addition, insertion or deletion) of one or more amino acid residues of the amino acid sequence.
  • the number of repeating units may be 1-20.
  • the number of repeating units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the mutation may be a substitution, such as a conservative amino acid substitution.
  • the amino acid sequence of SEQ ID NO.15 is gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak.
  • Each repeating unit may be directly connected or separated by one or more amino acid residues.
  • the obtained collagen or polypeptide retains the functions of the present invention, such as the ability of cell adhesion and cartilage repair.
  • the collagen or polypeptide of the present invention can be synthesized or can be expressed by recombinant expression.
  • the collagen or polypeptide of the present invention can be encoded by a polynucleotide.
  • the polynucleotide can be codon optimized for the host cell in which it is expressed.
  • the polynucleotide encoding the collagen or polypeptide can be operably connected to an expression control element, such as a promoter, a terminator and/or an enhancer to form a nucleic acid, or an expression cassette.
  • the nucleic acid can also include nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, or nucleotides encoding a leader sequence, to facilitate purification or secretion of the collagen or polypeptide.
  • a purification tag such as a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag
  • nucleotides encoding a leader sequence to facilitate purification or secretion of the collagen or polypeptide.
  • the term "vector” is a nucleic acid carrier into which a polynucleotide can be inserted.
  • a vector can express a protein encoded by the inserted polynucleotide
  • the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection to allow the genetic material it carries to be expressed.
  • the vector is a vector that is used to express a plasmid element in a host cell.
  • the vector is well known to those skilled in the art, and includes, but is not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • the vector may contain a variety of elements for controlling expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the vector may contain the nucleic acid of the present invention to facilitate introduction into cells for expression.
  • the vector may contain expression control elements operably linked to the nucleic acid, such as promoters, terminators and/or enhancers.
  • the term "host cell” is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • the host cell can be a eukaryotic cell or a prokaryotic cell.
  • a eukaryotic cell is a yeast cell, an animal cell, and/or an insect cell.
  • a prokaryotic cell can be an E. coli cell.
  • the present invention also provides a method for producing collagen or polypeptide, comprising: (1) culturing the host cell of the present invention under suitable culture conditions; (2) harvesting the host cell and/or culture medium containing the collagen or polypeptide; and (3) purifying the collagen or polypeptide.
  • the method of the present invention may include the step of cleaving the tag.
  • the collagen or polypeptide of the present invention can be prepared into a composition or a kit.
  • the composition or the kit can be a composition or a kit for tissue filling and/or volume expansion.
  • the composition or the kit can also contain auxiliary substances.
  • the composition of the present invention can be a cartilage repair agent, which contains the collagen or polypeptide described herein.
  • the composition of the present invention can be injectable.
  • the composition of the present invention can be a human structural material, for example, it can be used for cartilage repair, and does not cause an immune response in the human body.
  • the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably version 5.0.0 or later) is used to determine the sequence identity between two deoxynucleotide sequences.
  • the parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the non-simplified option) is used as the identity percentage and is calculated as follows:
  • the repeating unit of the collagen or polypeptide of the present invention or the collagen or polypeptide may have certain mutations.
  • the amino acid sequence of one or more of these parts may have substitution, deletion, addition or insertion of amino acid residues.
  • “multiple” can be 2-40, such as 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, 31, 32, 33, 34, 35, 36, 37, 38 or 39, or any numerical range therebetween.
  • the present invention can use repeat unit variants as long as the variant retains the activity of promoting cell adhesion.
  • the variant can have a certain percentage identity with the specified sequence (any collagen or polypeptide sequence described herein), 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 invention, such as SEQ ID NO.1-16, but it is preferred that these variants retain the intended function, such as increasing cell adhesion or cartilage repair.
  • the present invention provides a method for biosynthesizing recombinant humanized type II collagen, that is, a method for preparing human structural materials.
  • the method may include one or more of the following steps: (1) screening of functional regions and construction of bacterial strains; (2) large-scale biological fermentation and induced expression of proteins; (3) purification of humanized type II collagen and optional enzyme cleavage.
  • Functional region screening and strain construction can be carried out as follows: (1) large-scale functional region screening to obtain the functional region of the target gene; (2) inserting the obtained functional region of the target gene into the PET-28a-Trx-His expression vector to obtain a recombinant expression plasmid; (3) transferring the recombinant expression plasmid into the Escherichia coli competent cell BL21 (DE3) to screen and obtain positive Escherichia coli genetically engineered bacteria.
  • the induced expression of the protein can be carried out as follows: (1) cooling the shake flask after culture to 16-30°C; (2) adding IPTG mother solution to induce expression; (3) placing the bacterial solution after induced expression in a centrifuge bottle, centrifuging at 6000 rpm and 4°C for 12 minutes, and then collecting the bacteria.
  • the purification and optional enzymatic cleavage of humanized type II collagen can be carried out as follows: (1) crudely purifying humanized type II collagen on a Ni affinity chromatography column; (2) adding TEV enzyme for enzymatic cleavage at a certain ratio; (3) finely purifying humanized type II collagen on an ion exchange column.
  • the screened functional regions are as follows: (1) the amino acid sequence of HC2B-a3: gkpgddgeagkpgkagergppgpqgargfpgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagaagargndgqp; (2) the amino acid sequence of HC2B-a6: gkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkager
  • the amino acid sequence of the recombinant humanized type II collagen prepared by the present invention can be derived from the functional region of natural human type II collagen, including the functional region and similar functional regions, and proteins obtained by mutation and modification of the amino acid sequence.
  • the recombinant humanized type II collagen prepared by the present invention completely matches the amino acid sequence of human collagen, so it can be directly injected into the human body for cartilage repair without generating immunogenic reactions.
  • the present invention provides a collagen protein or polypeptide comprising an amino acid sequence of any one of SEQ ID NO.1-14 or an amino acid sequence in which the amino acid has been mutated.
  • the mutation may be a substitution, addition, deletion or insertion.
  • the substitution may be a conservative substitution.
  • the collagen protein or polypeptide of the present invention may be derived from a peptide segment derived from type II human collagen.
  • the collagen protein or polypeptide of the present invention may contain a plurality of peptide segments thus obtained.
  • the peptide segments may be connected via a linker or directly to form the collagen protein or polypeptide of the present invention.
  • the linker may be one or more amino acid residues.
  • the linker may be a flexible linker commonly used in the art.
  • the present invention provides a plurality of repeating units, as shown in the underlined amino acid sequences of the embodiments.
  • the present invention provides a repeating unit of the amino acid sequence of SEQ ID NO: 15.
  • the collagen or polypeptide of the present invention may comprise a plurality of repeating units, for example 2-30, 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 or 29.
  • Example 1 Construction and expression of recombinant humanized type II collagen
  • HC2B-b3 amino acid sequence gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO.4);
  • C (mg/ml) A280 ⁇ dilution factor ⁇ extinction coefficient)
  • electrophoresis detection e. Wash the column with 1 M imidazole working solution at a flow rate of 10 mL/min.
  • Enzyme digestion Add TEV enzyme at a ratio of 20:1 between the total amount of protein and the total amount of TEV enzyme, and digest at 16°C for 2 hours.
  • FIG. 1 shows the electrophoresis detection results of recombinant type II humanized collagen HC2B-a6 and HC2B-b5, showing that the expression level of the target protein after crude purification is low.
  • Figure 2 shows the electrophoresis detection results of recombinant type II humanized collagen HC2B-a3, HC2B-b1, and HC2B-b3, showing that there are many impurities after crude purification and fine purification of the protein.
  • Figure 3 shows the electrophoresis detection results of recombinant type II humanized collagen HC2B-A7 and HC2B-A8, showing that the expression level of the target protein after crude purification is low.
  • Figure 3 also shows the electrophoresis detection results of recombinant type II humanized collagen B3Q, showing that the amount of protein after fine purification is small.
  • Figure 4 shows the electrophoresis detection results of recombinant type II humanized collagen HC2B-A10, showing that the expression level of crude purified protein is high, the enzyme cutting effect is good, and the protein purity is high after fine purification (the left and middle figures of Figure 4).
  • Figures 4 and 7 also show the results of electrophoresis detection of recombinant type II humanized collagen HC2B-B9, showing that the expression of crude pure protein is low, there are many miscellaneous bands, and the enzyme cutting effect is poor.
  • Figure 5 shows the results of electrophoresis detection of recombinant type II humanized collagen HC2B-A9, showing that the expression of crude pure protein is high, there are many miscellaneous bands, and the purity is low.
  • the left figure of Figure 6 shows the results of electrophoresis detection of recombinant type II humanized collagen HC2B-B8, showing that the expression of crude pure protein is low, there are many miscellaneous bands, and no subsequent purification is performed.
  • the right figure of Figure 6 and Figure 7 show the results of electrophoresis detection of recombinant type II humanized collagen HC2B-B7, showing that the expression of crude pure protein is low, there are many miscellaneous bands, and the enzyme cutting effect is poor.
  • the middle figure of Figure 6 shows the electrophoresis detection of recombinant type II humanized collagen HC2B-B10, and the results show that the expression of crude pure protein is low, there are many miscellaneous bands, and no subsequent purification is performed.
  • recombinant type II humanized collagen HC2B-A10 achieves the best effect.
  • Example 2 Detection of biological activity of recombinant humanized type II collagen HC2B-A10
  • the activity detection method of collagen can refer to the literature 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 Collagen, J Biochem. 136, 643-649 (2004).
  • the specific implementation method is as follows:
  • 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 detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.). After the protein concentration is detected, the concentration of all proteins to be tested is adjusted to 0.5 mg/mL with PBS.
  • the cell adhesion rate can reflect the activity of collagen. The higher the activity of the protein, the better the external environment can be provided to the cells in a short time, helping the cells to adhere to the wall.
  • the results are shown in FIG8 . It can be seen from the comparison that the recombinant humanized type II collagen of the present invention has better bioadhesion activity than bovine type I collagen (B col I), that is, the recombinant humanized type II collagen HC2B-A10 > bovine type I collagen. Unexpectedly, the inventors have demonstrated that for HC2B-A10, when the number of repeating sequences is 8, the molecular weight of the recombinant humanized type II collagen can be easily purified and stabilized, and has excellent bioadhesion activity.
  • Example 3 Mass spectrometry detection of recombinant humanized type II collagen HC2B-A10
  • the recombinant type II humanized collagen HC2B-A10 with good purification and enzyme cleavage effects provided by the present invention is subjected to mass spectrometry detection, and the specific process is as follows:
  • Protein identification results were obtained based on the primary mass spectra of the peptides produced after enzymatic digestion. Detection parameters: Trypsin digestion, two missed cleavage sites. Alkylation of cysteine was set as a fixed modification. Oxidation of methionine was set as a variable modification.
  • the database used for identification was NCBprot.
  • Table 1 Molecular weights and corresponding peptides detected by HC2B-A10 mass spectrometry
  • the coverage of the detected peptide functional regions is 100%

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Abstract

提供了胶原蛋白及其用途。胶原蛋白包含N端序列和C端序列，其中N端序列包含一个或多个重复单元，该重复单元包含以SEQ ID NO.15所示的氨基酸序列。本发明制备得到的重组Ⅱ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，可大规模获得重组Ⅱ型人源化胶原蛋白，并在软骨修复领域有望得到广泛的应用。

Description

胶原蛋白及其用途
本申请要求申请日：2023年5月12日，申请号：202310537499.9，发明名称“多肽及其用途”的中国发明专利申请的优先权权益。上述申请的内容通过引用并入本文。
技术领域
本发明属于合成生物技术领域，并且具体涉及人体结构性材料以及其生物合成制备方法。
背景技术
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。
Ⅱ型胶原蛋白主要存在于软骨组织、玻璃体和眼角膜中，是一种高分子蛋白质，其丝状胶原蛋白纤维与弹性蛋白及多糖蛋白相互交织形成网状结构，又称复合骨胶原。Ⅱ型胶原蛋白是软骨和骨型形成、骨骼生长和成熟软骨维持等所必需的成分，所以非变性的胶原蛋白可作为软骨的一种结构功能性成分。
Ⅱ型胶原蛋白作为关节软骨基质的主要组成成分，与透明质酸和蛋白多糖等润滑成分共同保护软骨，免于磨损。随着年龄的增加，胶原蛋白的合成速度逐渐降低，胶原蛋白的流失加剧，关节软骨退变，骨与骨之间的摩擦加剧，会引发关节处炎症发生。目前对骨关节炎常见的治疗方法是食用胶原蛋白，但是其蛋白利用率低，导致软骨再生的速度缓慢，不利于病人的康复，并影响治疗的进程和效率。因目前的治疗手段疗效不理想，往往患者最终不得不选择人工关节置换术，不仅花费大，而且可能存在术后血栓形成，置换术后感染以致截肢等灾难性并发症。
然而，目前用作软骨的胶原蛋白材料主要源自于动物软骨提取，动物源胶原蛋白的免疫源性无法消除，其除杂和提取过程仍然较为复杂，很难保障胶原蛋白的三螺旋结构，无法用于大规模生产。
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功能区，或是含人胶原蛋白功能的功能区的组合。
生产胶原蛋白的传统方法是利用酸、碱、酶解法处理动物来源的组织，提取胶原蛋白衍生物，这些方法提取的胶原蛋白已经丧失了原本的生物学活性，无法应用于生物医学领域发挥真正的功能。随着现代技术的发展，国内外出现了一些对动物软骨进行除杂和酶解提取得到非变性Ⅱ型胶原蛋白的提取方法。虽其制备得到的胶原蛋白可以应用于软骨修复，但是该方法除杂和提取的时间长，产品纯度低，产品稳定性差，不利于规模生产。针对现有技术的缺陷，有公司提出采用毕赤酵母菌作为宿主菌株，制备得到Ⅱ型类胶原蛋白以用于软骨修复，但是该胶原蛋白并不属于人源化胶原蛋白，所以具有一定的免疫原性反应。
因此，亟需一种可直接注射于人体，且不会引起免疫原性反应的重组Ⅱ型人源化胶原蛋白，以作为人体结构材料应用于软骨修复。
发明内容
人体结构性材料主要是包含胶原蛋白之内的结构性蛋白，这类蛋白结构复杂、功能精确，对人体组织修复再生有重要意义，但用常规生产方法不容易获得。本发明运用合成生物与结构生物学技术，开发了一种具有人Ⅱ型胶原蛋白三螺旋结构、并能行使人胶原蛋白功能的一种大分子功能蛋白。发明人进行了大规模功能区筛选，一是序列筛选，在天然Ⅱ型胶原蛋白螺旋区的Gly-X-Y的重复基因序列上含有大量的带电氨基酸，这些电荷通过相互作用来结合细胞，因此排除不含有这些重要电荷基序的区域；二是借助计算机辅助蛋白结构预测的方法来帮助筛选出最多链间氢键结构、最能稳定三聚体聚集形式的潜在螺旋功能区；三是依据蛋白质表达性质的预测方法，筛选出蛋白表达量最高，易于纯化，稳定性好的人Ⅱ型胶原蛋白功能区域；四是将这些区域的氨基酸片段通过n次重复(重复是为了保证重组Ⅱ型人源化胶原蛋白的分子量在一定范围易于纯化和稳定)直接连接进行优化。
本发明包含了重组Ⅱ型人源化胶原蛋白的核心功能区筛选、合成工艺及 应用场景。本专利设计首次发明了一种直接注射人体的重组Ⅱ型人源化胶原蛋白的功能区筛选和蛋白合成工艺，可用于软骨修复。
本发明部分基于发明人如下的意料不到的发现。与其他重组Ⅱ型人源化胶原蛋白相比，重组Ⅱ型人源化胶原蛋白HC2B-A10在重组表达时表达量更大，更易于分离和纯化，并且与牛Ⅰ型胶原蛋白相比具有优异的细胞粘附效果。
在一方面，本发明提供了胶原蛋白或多肽，包含多个重复单元，该重复单元包含以SEQ ID NO.15所示的氨基酸序列(gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak)或与其具有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的变体，各重复单元是直接连接的或间隔一个或多个氨基酸残基。在一个实施方案中，重复单元可以是SEQ ID NO.15的氨基酸序列经过一个或多个氨基酸残基的突变(取代、插入、删除或添加)后得到的氨基酸序列。
在一个实施方案中，重复单元的数目是1-20。在一个实施方案中，重复单元的数目是6-10。在一个实施方案中，重复单元的数目是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。
在一个实施方案中，胶原蛋白或多肽包含以SEQ ID NO.10所示的氨基酸序列。在一个实施方案中，胶原蛋白或多肽包含SEQ ID NO.10所示的氨基酸序列或SEQ ID NO.3或10的氨基酸序列经过一个或多个氨基酸残基的突变(取代、插入、删除或添加)后得到的氨基酸序列或与SEQ ID NO.10所示的氨基酸序列具有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的变体。在一个实施方案中，在胶原蛋白或多肽序列发生突变的情况下，得到的胶原蛋白或多肽保留本发明的功能，如细胞粘附、软骨修复的能力等。
在一方面，本发明提供了多核苷酸，其编码本文所述的胶原蛋白或多肽。
在一个实施方案中，多核苷酸包含SEQ ID NO.29所示的核苷酸序列。
在一方面，本发明提供了核酸，其包含本文所述的多核苷酸。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。
在一方面，本发明提供了载体，其包含本文所述的多核苷酸或本文所述的核酸。
在一个实施方案中，载体是表达载体。在一个实施方案中，载体包含与多核苷酸或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。
在一方面，本发明提供了宿主细胞，其包含本文所述的多核苷酸、本文所述的核酸或本文所述的载体。在一个实施方案中，宿主细胞是细菌、真菌或动物细胞。在一个实施方案中，细菌是大肠杆菌。在一个实施方案中，真菌是酵母，例如酿酒酵母。
在一方面，本发明提供了生产本文所述的胶原蛋白或多肽的方法，其包括：
(1)在合适的培养条件下培养本文所述的宿主细胞；
(2)收获包含胶原蛋白或多肽的宿主细胞和/或培养基；和
(3)纯化胶原蛋白或多肽。
在一方面，本发明提供了组合物，其包含本文所述的胶原蛋白或多肽。在一个实施方案中，组合物是试剂盒。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。在一个实施方案中，组合物为注射用组合物或口服用组合物。
在一个实施方案中，组合物是用于软骨修复的组合物。在一个实施方案中，组合物是用于软骨修复的注射用组合物。
在一方面，本发明提供了用于增加细胞粘附的方法，其包括将细胞与本文所述的胶原蛋白或多肽、本文所述的多核苷酸、本文所述的核酸、本文所述的载体、本文所述的宿主细胞、和/或本文所述的组合物接触。
在一方面，本发明提供了本文所述的胶原蛋白或多肽、本文所述的多核苷酸、本文所述的核酸、本文所述的载体、本文所述的宿主细胞、和/或本文所述的组合物在制备试剂盒中的用途，所述试剂盒用于增加细胞粘附或软骨修复。在一方面，本发明提供了本文所述的胶原蛋白或多肽、本文所述的多核苷酸、本文所述的核酸、本文所述的载体、本文所述的宿主细胞、和/或本文所述的组合物在生物敷料、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中的用途。
本发明的优点包括：
1.本发明提供了重组Ⅱ型人源化胶原蛋白的核心功能区及氨基酸序列。
2.本发明首次成功合成了可注射于人体的重组Ⅱ型人源化胶原蛋白，用于软骨修复。
3.生产的重组Ⅱ型人源化胶原蛋白其氨基酸组成与天然胶原蛋白氨基酸序列相应部分100％相同，应用于人体不会产生免疫排斥和过敏反应。
4.本文的制备方法简单，可大规模生产高产量的重组Ⅱ型人源化胶原蛋白。
5.就表达、分离和纯化效果而言，与其他重组II型人源化胶原蛋白相比，重组II型人源化胶原蛋白HC2B-A10实现最佳的效果。
6.本发明的重组Ⅱ型人源化胶原蛋白具有更加优秀的生物黏附活性，即重组Ⅱ型人源化胶原蛋白HC2B-A10>牛Ⅰ型胶原蛋白。
附图说明
图1：重组Ⅱ型人源化胶原蛋白HC2B-a6和HC2B-b5的粗纯情况；
图2：重组Ⅱ型人源化胶原蛋白HC2B-b1、HC2B-a3和HC2B-b3的粗纯情况；
图3：重组Ⅱ型人源化胶原蛋白B3Q、HC2B-A8和HC2B-A7的纯化情况；
图4：重组Ⅱ型人源化胶原蛋HC2B-A10的纯化情况和HC2B-B9的粗纯情况；
图5：重组Ⅱ型人源化胶原蛋白HC2B-A9的纯化情况；
图6：重组Ⅱ型人源化胶原蛋白HC2B-B8、HC2B-B7和HC2B-B10的粗纯情况；
图7：重组Ⅱ型人源化胶原蛋白HC2B-B7和HC2B-B9的精纯情况；
图8：重组Ⅱ型人源化胶原蛋白HC2B-A10的活性检测情况；
图9：pET-28a-Trx-His表达载体图谱。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明的实施例，对本发明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发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，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，都属于本发明保护的范围。
重组胶原蛋白是利用前沿的结构生物学、基因工程等技术，以人特定型别胶原蛋白功能区基因编码为模板，进行筛选、制备得到与人胶原蛋白氨基酸序列相同或类似的一类新型生物材料。
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，胶原蛋白、重组胶原蛋白、重组Ⅱ型人源化胶原蛋白或多肽之间可以互换使用。
在本文中，胶原蛋白或多肽可以包含一个或多个重复单元，该重复单元包含以SEQ ID NO.15所示的氨基酸序列或者该氨基酸序列经过一个或多个氨基酸残基的突变(取代、添加、插入或缺失)后的氨基酸序列。重复单元的数目可以是1-20。例如，重复单元的数目是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。特别地，突变可以是取代，例如保守氨基酸取代。SEQ ID NO.15的氨基酸序列为gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak。各重复单元可以是直接连接的或间隔一个或多个氨基酸残基。
在胶原蛋白或多肽序列发生突变或存在间隔序列的情况下，得到的胶原蛋白或多肽保留本发明的功能，如细胞粘附、软骨修复的能力等。
本发明的胶原蛋白或多肽可以是合成的或者可以通过重组表达。在重组表达的情况下，本发明的胶原蛋白或多肽可以由多核苷酸编码。多核苷酸可以针对进行表达的宿主细胞进行密码子优化。编码胶原蛋白或多肽的多核苷酸可以与表达控制元件，如启动子、终止子和/或增强子可操作连接以构成核酸，或者表达盒。该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标签的核苷酸或者编码前导序列的核苷酸，以便于胶原蛋白或多肽的纯化或分泌。
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。载体可以包含本发明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。
本发明还提供了生产胶原蛋白或多肽的方法，其包括：(1)在合适的培养条件下培养本文的宿主细胞；(2)收获包含胶原蛋白或多肽的宿主细胞和/或培养基；和(3)纯化胶原蛋白或多肽。本发明的方法可以包括酶切标签的步骤。
本发明的胶原蛋白或多肽可以制备成组合物或试剂盒。组合物或试剂盒可以是组织填充和/或增容用的组合物或试剂盒。组合物或试剂盒还可以包含辅助物质。本发明的组合物可以是软骨修复剂，其包含本文所述的胶原蛋白或多肽。本发明的组合物可以是可注射的。本发明的组合物可以是人体结构性材料，例如可用于软骨修复，且不引起人体的免疫应答。
如本文中所用，两个氨基酸序列之间或两个核苷酸序列之间的关联度通过参数“序列同一性”来描述。出于本发明的目的，使用如在EMBOSS软件包(EMBOSS：欧洲分子生物学开放软件套件，Rice等人，2000,Trends Genet.[遗传学趋势]16:276-277)(优选5.0.0版或更新版本)的尼德尔程序所实施的尼德曼-翁施算法(Needleman和Wunsch，1970,J.Mol.Biol.[分子生物学杂志]48:443-453)来确定两个氨基酸序列之间的序列同一性。使用的参数是空位开放罚分10、空位延伸罚分0.5以及EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。将标记为“最长同一性”的尼德尔的输出(使用非简化选项获得)用作同一性百分比并且计算如下：
(相同的残基×100)/(比对长度-比对中的空位总数)
出于本发明的目的，使用如在EMBOSS软件包(EMBOSS：欧洲分子生物学开放软件套件，Rice等人,2000,同上)(优选5.0.0版本或更新版本)的尼德尔程序所实施的尼德曼-翁施算法(Needleman和Wunsch,1970,同上)来确定两个脱氧核苷酸序列之间的序列同一性。所使用的参数是空位开放罚分10、空位延伸罚分0.5、和EDNAFULL(NCBI NUC4.4的EMBOSS版)取代矩阵。将标记为“最长同一性”的尼德尔的输出(使用非简化选项获得)用作同一性百分比并且计算如下：
(相同的脱氧核糖核苷酸x 100)/(比对长度-比对中的空位总数)
在本文中，本发明的胶原蛋白或多肽的重复单元或胶原蛋白或多肽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。在氨基酸突变的上下文中，“多个”可以是2-40，例如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、31、32、33、34、35、36、37、38或39，或者它们之间的任何数值范围。
也就是说，本发明可以使用重复单元变体，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(本文中所述的任何胶原蛋白或多肽序列)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发明的任何序列，例如SEQ ID NO.1-16，但是优选的是这些变体保留意图的功能，例如增加细胞粘附或软骨修复。
本发明的方法
针对目前的研究现状，本发明提供了一种生物合成重组Ⅱ型人源化胶原蛋白的方法，即人体结构性材料的制备方法。方法可以包括以下一个或多个步骤：(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Ⅱ型胶原蛋白的纯化和任选的酶切。
功能区筛选和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-28a-Trx-His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。
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。
蛋白的诱导表达可如下进行：(1)将培养后的摇瓶降温至16-30℃；(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，6000rpm、4℃离心12min后收集菌体。
人源化Ⅱ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Ⅱ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Ⅱ型胶原蛋白。
筛选出的功能区如下所示：(1)HC2B-a3的氨基酸序列：gkpgddgeagkpgkagergppgpqgargfpgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagaagargndgqp；(2)HC2B-a6的氨基酸序列：gkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagerg；(3)HC2B-b1的氨基酸序列：gepgregspgadgppgrdgaagvkgdrgetgavgapgapgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgppgpvgpsgkdgangipgpigppgprgrsgetgpa；(4)HC2B-b3氨基酸序列：gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr；(5)HC2B-b5的氨基酸序列：gargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgps；(6)B3Q的氨基酸序列：gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpa gpsgpr；(7)HC2B-A7的氨基酸序列：gpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgak；(8)HC2B-A8的氨基酸序列：gargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgak；(9)HC2B-A9的氨基酸序列：gfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgak：(10)HC2B-A10的氨基酸序列：gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak；(11)HC2B-B7的氨基酸序列：gerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr；(12)HC2B-B8的氨基酸序列：gerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppg psgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpa；(13)HC2B-B9的氨基酸序列：gerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdq；(14)HC2B-B10的氨基酸序列：gerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgpp。
本发明制备的重组Ⅱ型人源化胶原蛋白氨基酸序列可以源自人天然Ⅱ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。
本发明制备得到的重组Ⅱ型人源化胶原蛋白完全匹配人体胶原蛋白的氨基酸序列，所以可直接注射人体用于软骨修复而不产生免疫原性反应。本发明的胶原蛋白或多肽
本发明提供了胶原蛋白或多肽，其包含SEQ ID NO.1-14中任一项的氨基酸序列或者该氨基酸经过突变的氨基酸序列。突变可以是取代、添加、缺失或插入。优选地，取代可以是保留的取代。本发明的胶原蛋白或多肽可以衍生自从Ⅱ型人胶原蛋白衍生得到的肽段。本发明的胶原蛋白或多肽可以包含多个如此得到的肽段。肽段可以通过接头或直接连接形成本发明的胶原蛋白或多肽。接头可以是一个或多个氨基酸残基。例如，接头可以是本领域中通常使用的柔性接头。
本发明提供了多个重复单元，如实施例的下划线表示的氨基酸序列所示。特别地，本发明提供了SEQ ID NO:15的氨基酸序列的重复单元。本发明的胶原蛋白或多肽可以包含多个重复单元，例如2-30，2、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或29。
实施例
提供以下实施例来阐述本发明。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。本发明仅仅由所附权利要求书的范围限定。
实施例1：重组Ⅱ型人源化胶原蛋白构建及表达
1.进行大规模功能区筛选，一是序列筛选，在天然Ⅱ型胶原蛋白螺旋区的Gly-X-Y的重复基因序列上含有大量的带电氨基酸，这些电荷通过相互作用来结合细胞，因此排除不含有这些重要电荷基序的区域；二是借助计算机辅助蛋白结构预测的方法来帮助筛选出最多链间氢键结构、最能稳定三聚体聚集形式的潜在螺旋功能区；三是依据蛋白质表达性质的预测方法，筛选出蛋白表达量最高，易于纯化，稳定性好的人Ⅱ型胶原蛋白功能区域；四是将这些区域的氨基酸片段通过n次重复(重复是为了保证重组Ⅱ型人源化胶原蛋白的分子量在一定范围易于纯化和稳定)直接连接进行优化；得到以下不同的重组Ⅱ型人源化胶原蛋白的目的基因功能区：
(1)HC2B-a3的氨基酸序列：gkpgddgeagkpgkagergppgpqgargfpgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagaagargndgqp(SEQ ID NO.1)；
(2)HC2B-a6的氨基酸序列：gkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagerg(SEO ID NO.2，下划线为重复单元部分)；
(3)HC2B-b1的氨基酸序列：gepgregspgadgppgrdgaagvkgdrgetgavgapgapgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgppgpvgpsgkdgangipgpigppgprgrsgetgpa(SEQ ID NO.3)；
(4)HC2B-b3氨基酸序列：gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr(SEQ ID NO.4)；
(5)HC2B-b5的氨基酸序列：gargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglq glpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgps(SEQ ID NO.5，下划线为重复单元部分)；
(6)B3Q的氨基酸序列：gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerGlkghrgftgl qglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr(SEQ ID NO.6，下划线为重复单元部分)；
(7)HC2B-A7的氨基酸序列：gpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgak(SEQ ID NO.7，下划线为重复单元部分)；
(8)HC2B-A8的氨基酸序列：gargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgak(SEQ ID NO.8，下划线为重复单元部分)；
(9)HC2B-A9的氨基酸序列：gfpgtpglpgvkghrgypgldgakgfpgtpglpgVkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgak(SEQ ID NO.9，下划线为重复单元部分)；
(10)HC2B-A10的氨基酸序列： gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak
gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak(SEQ ID NO.10)；
(11)HC2B-B7的氨基酸序列：gerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr(SEQ ID NO.11，下划线为重复单元部分)；
(12)HC2B-B8的氨基酸序列：gerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpa(SEQ ID NO.12)；
(13)HC2B-B9的氨基酸序列：gerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdq(SEQ ID NO.13)；
(14)HC2B-B10的氨基酸序列：gerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkg hrgftglqglpgppgerglkghrgftglqglpgpp(SEQ ID NO.14)。
2.将合成的基因功能区插入pET-28a-Trx-His表达载体(如图9)中得到对应的重组表达质粒。
3.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硫酸卡那霉素(50mg/L)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。
4.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(氨苄青霉素100mg/L)的摇瓶中，在220rpm，37℃恒温摇床中培养一定时间，直至其呈现雾状。再将培养后的摇瓶降温至16-30℃，添加IPTG(0.5mM)诱导表达一段时间后，将菌液分装于离心瓶中，于6000rpm、4℃离心12min，收集菌体，并记录菌体重量，取样进行电泳检测。
5.将收集的菌体用平衡工作液(200mM氯化钠，25mM Tris，20mM咪唑)重悬，将菌液降温至≤15℃，进行均质两次或超声细胞破碎，完成后收集菌液。将细胞破碎后的菌液分装至离心瓶中，于17000rpm、4℃离心30min，收集上清液。
6.将重组人源化Ⅱ型胶原蛋白的进行纯化和酶切，具体过程是：(1)粗纯：a.平衡柱材：使用平衡液(200mM氯化钠，25mM Tris，20mM咪唑)将柱材平衡，流速为10mL/min。b.上样：将离心后的上清液加入柱材中，直至液体流完后，流速为5mL/min。c.清洗杂蛋白：添加洗杂液100mL(200mM氯化钠，25mM Tris，20mM咪唑)至液体流完，流速为10mL/min。d.收集目的蛋白：添加20mL洗脱液(200mM氯化钠，25mM Tris，250mM咪唑)，流速为10mL/min，并收集流穿液，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)。e.用1M咪唑工作液清洗柱材，流速为10mL/min。(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。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(20mM氯化钠，20mM  Tris)中4℃过夜透析。(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，上样并收集流穿样品，并进行电泳检测，将蛋白储存在4℃环境中。c.洗脱：用B液(1M氯化钠，20mM Tris)清洗柱材5个CV。d.清洗柱材。
试验结果：
各重组蛋白在制备过程中所涉及到的电泳检测结果见图1-图7。图1显示了重组II型人源化胶原蛋白HC2B-a6、HC2B-b5电泳检测结果，显示了粗纯后目的蛋白表达量较少。图2显示了重组II型人源化胶原蛋白HC2B-a3、HC2B-b1、HC2B-b3电泳检测结果，显示了蛋白粗纯和精纯后杂蛋白较多。图3显示了重组II型人源化胶原蛋白HC2B-A7、HC2B-A8电泳检测结果，显示了粗纯后目的蛋白表达量较少。图3还显示了重组II型人源化胶原蛋白B3Q电泳检测结果，显示了精纯后蛋白量较少。图4显示了重组II型人源化胶原蛋白HC2B-A10电泳检测结果，显示了粗纯蛋白表达量较高，酶切效果较好，精纯后蛋白纯度高(图4的左侧和中间图)。图4和图7还显示了重组II型人源化胶原蛋白HC2B-B9电泳检测结果，显示了粗纯蛋白表达量较少，且杂带较多，酶切效果较差。图5显示了重组II型人源化胶原蛋白HC2B-A9电泳检测结果，显示了粗纯蛋白表达量较高，杂带较多，纯度较低。图6的左图显示了重组II型人源化胶原蛋白HC2B-B8电泳检测结果，显示了粗纯蛋白表达量较少，杂带较多，不再进行后续精纯。图6的右图和图7显示了重组II型人源化胶原蛋白HC2B-B7电泳检测结果，显示了粗纯蛋白表达量较少，杂带较多，酶切效果较差。图6的中间图显示了重组II型人源化胶原蛋白HC2B-B10电泳检测，结果显示了粗纯蛋白表达量较少，杂带较多，不再进行后续精纯。
因此，就表达、分离和纯化效果而言，与其他重组II型人源化胶原蛋白相比，重组II型人源化胶原蛋白HC2B-A10实现最佳的效果。
实施例2：重组Ⅱ型人源化胶原蛋白HC2B-A10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型胶原标准品 (Sigma，编号：380002)、本发明提供的纯化和酶切效果较好的重组Ⅱ型人源化胶原蛋白HC2B-A10(编号为015)。
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0.5mg/mL。
(2)向96孔板中加入100μL各种蛋白溶液和空白PBS溶液对照，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8所示，从对比中可知，相比于牛Ⅰ型胶原蛋白(B col I)，本发明的重组Ⅱ型人源化胶原蛋白具有更加优秀的生物黏附活性，即重组Ⅱ型人源化胶原蛋白HC2B-A10>牛Ⅰ型胶原蛋白。出乎意料地，本发明人证明了对于HC2B-A10，重复序列在8个时可以保证重组Ⅱ型人源化胶原蛋白的分子量易于纯化和稳定，且具有优秀的生物黏附活性。
实施例3：重组Ⅱ型人源化胶原蛋白HC2B-A10的质谱检测
实验方法
将本发明提供的纯化和酶切效果较好的重组Ⅱ型人源化胶原蛋白HC2B-A10进行质谱检测，具体过程如下：
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：HC2B-A10质谱检出分子量及对应多肽
检出多肽功能区的覆盖率为100％

HC2B-a3的DNA序列(SEQ ID NO.16)：
HC2B-a6的DNA序列(SEQ ID NO.17)：
HC2B-b1的DNA序列(SEQ ID NO.18)：
HC2B-b3的DNA序列(SEQ ID NO.19)：
HC2B-b5的DNA序列(SEQ ID NO.20)：

B3Q的DNA序列(SEQ ID NO.21)：
HC2B-A7的DNA序列(SEQ ID NO.22)：

HC2B-A8的DNA序列(SEQ ID NO.23)：
HC2B-A9的DNA序列(SEQ ID NO.24)：
HC2B-A10的DNA序列(SEQ ID NO.25)：

HC2B-B7的DNA序列(SEQ ID NO.26)：
HC2B-B8的DNA序列(SEQ ID NO.27)：

HC2B-B9的DNA序列(SEQ ID NO.28)：
HC2B-B10的DNA序列(SEQ ID NO.29)：

Claims (10)

  1. 胶原蛋白，包含多个重复单元，该重复单元包含以SEQ ID NO.15所示的氨基酸序列、SEQ ID NO.15的氨基酸序列经过一个或多个氨基酸残基的突变(取代、插入、删除或添加)后得到的氨基酸序列或与其具有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列，各重复单元是直接连接的或间隔一个或多个氨基酸残基；优选地，其中重复单元的数目是1-20；优选地重复单元的数目是6-10，例如7、8或9；优选地，胶原蛋白包含以SEQ ID NO.10所示的氨基酸序列、SEQ ID NO.10的氨基酸序列经过一个或多个氨基酸残基的突变(取代、插入、删除或添加)后得到的氨基酸序列或与其具有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的变体；优选地，胶原蛋白是重组Ⅱ型人源化胶原蛋白。
  2. 多核苷酸，其编码根据权利要求1所述的胶原蛋白；优选地，所述多核苷酸包含SEQ ID NO.29所示的核苷酸序列。
  3. 核酸，其包含根据权利要求2所述的多核苷酸，任选地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，任选地还包含编码前导序列的核苷酸。
  4. 载体，其包含根据权利要求2所述的多核苷酸或根据权利要求3所述的核酸，优选地，其中载体是表达载体，优选地，其中载体包含与多核苷酸或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。
  5. 宿主细胞，其包含根据权利要求2所述的多核苷酸、根据权利要求3所述的核酸或根据权利要求4所述载体；优选地，其中宿主细胞是细菌、真菌或动物细胞，优选地，细菌是大肠杆菌；优选地，真菌是酵母，例如酿酒酵母。
  6. 生产根据权利要求1所述的胶原蛋白的方法，其包括：
    (1)在合适的培养条件下培养根据权利要求5所述的宿主细胞；
    (2)收获包含胶原蛋白的宿主细胞和/或培养基；和
    (3)纯化胶原蛋白。
  7. 组合物，其包含根据权利要求1所述的胶原蛋白，优选地，组合物是试剂盒；优选地，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，优选地其中组合物为注射用组合物或口服用组合物。
  8. 根据权利要求7所述的组合物，其是用于软骨修复的组合物，优选是用于软骨修复的注射用组合物。
  9. 用于增加细胞粘附的方法，其包括将细胞与根据权利要求1所述的胶原蛋白、根据权利要求2所述的多核苷酸、根据权利要求3所述的核酸、根据权利要求4所述的载体、根据权利要求5所述的宿主细胞、和/或根据权利要求7或8所述的组合物接触。
  10. 根据权利要求1所述的胶原蛋白、根据权利要求2所述的多核苷酸、根据权利要求3所述的核酸、根据权利要求4所述的载体、根据权利要求5所述的宿主细胞、和/或根据权利要求7或8所述的组合物在制备试剂盒中的用途，所述试剂盒用于增加细胞粘附或软骨修复，或者在生物敷料、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中的用途。
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