WO2021083072A1 - 人胶原蛋白17型多肽、其生产方法和用途 - Google Patents

人胶原蛋白17型多肽、其生产方法和用途 Download PDF

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WO2021083072A1
WO2021083072A1 PCT/CN2020/123600 CN2020123600W WO2021083072A1 WO 2021083072 A1 WO2021083072 A1 WO 2021083072A1 CN 2020123600 W CN2020123600 W CN 2020123600W WO 2021083072 A1 WO2021083072 A1 WO 2021083072A1
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seq
polypeptide
amino acid
protein
collagen
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PCT/CN2020/123600
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English (en)
French (fr)
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朱赟
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山西锦波生物医药股份有限公司
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Priority to BR112022004900-5A priority Critical patent/BR112022004900B1/pt
Priority to US17/761,992 priority patent/US12043657B2/en
Priority to KR1020227011669A priority patent/KR102665118B1/ko
Priority to JP2022518672A priority patent/JP7300060B2/ja
Priority to EP20881368.3A priority patent/EP4053160A4/en
Publication of WO2021083072A1 publication Critical patent/WO2021083072A1/zh
Priority to US18/612,612 priority patent/US20240309067A1/en
Priority to US18/612,627 priority patent/US20240301035A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • A23L29/284—Gelatin; Collagen
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
    • 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
    • A61K8/65—Collagen; Gelatin; Keratin; 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
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • 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
    • 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
    • A61Q19/08—Anti-ageing preparations
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of genetic engineering and relates to polypeptides, production methods and uses thereof.
  • Collagen is generally white, transparent, and unbranched fibrils. It is the basic support for skin and bones. It can account for 25% to 35% of the total protein. It is mainly distributed in the skin, blood vessels, bones, tendons, and tendons of the human body. Teeth and cartilage are the main matrix and scaffolds of these tissues, which protect and connect various tissues and play important physiological functions in the body. Therefore, collagen can be widely used in industries such as medicine and cosmetics.
  • the collagen products currently on the market are all taken from animal tissues such as pigs, cattle, and fish. Although the collagen of some animals and humans is highly similar, it is still difficult to avoid the risk of viral infection and sensitization. At present, a small amount of animal-derived collagen has been used in cosmetics, but it is difficult to be widely used in medical equipment or more sophisticated tissue engineering products, and it cannot play the original biological function of collagen at all. Moreover, collagen prepared by conventional methods generally has a strong coagulation function, which leads to a great risk of thrombosis when it is used in certain tissue engineering products, which greatly limits its extensive and in-depth application.
  • the traditional method of producing collagen is to use acid, alkali, and enzymatic hydrolysis to treat tissues derived from animals to extract collagen derivatives.
  • the collagen extracted by these methods has lost its original biological activity and cannot be used in the field of biomedicine to perform its real function.
  • Some research institutions at home and abroad express human collagen in vitro through conventional recombinant expression methods, but the production cost is usually too high and the production cycle is too long to be put into large-scale production. Therefore, there is an urgent need in the market for a collagen material with excellent biomaterial properties, a high degree of amino acid sequence homology with the human body, and which can be produced in large quantities in an industrial system.
  • the most common structural feature of collagen is the triple helix structure formed by three peptide chains, that is, the three A peptide chains form a protein in a right-handed supercoiled manner. Such a triple helix area is called a collagen area.
  • each A peptide chain is composed of repeated Gly-XY (X and Y represent any amino acid residues other than Gly, X is often Pro, Y is often Hyp) peptides constituting a left-hand helix, 3
  • X is often Pro
  • Y is often Hyp
  • the chain is centered on the same axis and forms a stable triple helix structure in a right-handed supercoiled manner. Therefore, it is difficult for ordinary collagen sequences to spontaneously combine to form a stable triple helix structure to perform biological functions. Such difficulties severely hinder the development and production of human collagen.
  • the human body contains 28 different types of collagen, which are divided into common fibrous collagen and uncommon non-fibrous collagen.
  • Type I, type II, and type III of human skin belong to fibrous collagen.
  • a very important subtype of collagen is type 17 collagen collagen XVII (encoded by the COL17A1 gene in the human body).
  • Type 17 collagen is a homotrimer formed by the combination of three COL17A1 chains, with a single chain molecular weight of 180kDa. It contains a spherical intracellular domain of 70kDa, a transmembrane domain and an extracellular collagen domain of 120kDa, which have very strong thermal stability.
  • Recent studies have confirmed that type 17 collagen is an important component of epidermal stem cells in the human body and plays an important role in cell aging and skin differentiation.
  • humans currently have very limited understanding of the structure and function of non-fibrous collagen, especially for type 17 collagen.
  • the inventor has studied the structure and function of collagen in depth for many years. In particular, for the first time in the world, he analyzed the new atomic structure of multiple segments of human collagen, and posted it to the international protein structure database for public display, and accumulated rich research experience. . Through repeated explorations, the inventors have successfully achieved the recombinant expression of several extracellular functional regions of type 17 collagen, and found that it has excellent biomaterial properties, its preparation method is simple, easy to expand production, and can be widely used in medicine and cosmetics, etc. Industry.
  • the present invention is based in part on the following findings:
  • polypeptides C17A3, C17B3 and C17C1 of the present invention have comparable or higher cell adhesion effects compared with existing human collagen, and the polypeptides C17A3, C17B3 and C17C1 exist in water-soluble form after being expressed in host cells, and the preparation method is simple, Easy to expand production.
  • the present invention provides:
  • Item 1 A polypeptide comprising 63 to 1496 consecutive amino acid residues in SEQ ID No. 9, wherein the polypeptide has cell adhesion activity.
  • polypeptide wherein the polypeptide comprises the sequence shown in (A) m or consists of the sequence shown in (A) m , wherein each A is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQ
  • the amino acid sequence shown in any one of ID No. 3 or any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 has one or more substitutions, additions, or deletions, such as 2, 3, 4 or The amino acid sequence of 5 amino acid residues or the amino acid sequence shown in any one of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.
  • m is an integer between 1-10; where each A is the same Or different and adjacent two A are directly connected by a peptide bond or connected by more than one amino acid residue; wherein the polypeptide has cell adhesion activity.
  • the interval described herein includes endpoints, for example, between 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, that is, m may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • polypeptide of item 1 or 2 wherein the polypeptide comprises the amino acids shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 6 Sequence or consist of it.
  • polynucleotide which encodes the polypeptide according to any one of items 1-3, preferably, the polynucleotide comprises SEQ ID No. 5, SEQ ID No. 7, or SEQ ID No. 8
  • the nucleotide sequence shown may consist of the nucleotide sequence shown in SEQ ID No. 5, SEQ ID No. 7, or SEQ ID No. 8.
  • Item 6 A host cell comprising the expression vector according to item 5 or the polypeptide according to any one of items 1 to 3, wherein the host cell is preferably an E. coli cell.
  • Item 7 A method for preparing the polypeptide according to any one of items 1-3, which comprises:
  • Item 8 A composition comprising the polypeptide according to any one of items 1 to 3 or the polypeptide prepared according to the method of item 7.
  • a product comprising the polypeptide according to any one of items 1-3 or the polypeptide prepared according to the method of item 7 or the composition according to item 8, wherein the finished product is a pharmaceutical composition or a medical device , Tissue engineering products, cosmetics or health products, preferably the pharmaceutical composition is an external preparation, preferably an external smear preparation, such as an external gel or an external infiltration preparation; wherein preferably the external gel also contains a pharmaceutically acceptable
  • the accepted carrier, the external infiltration preparation also contains a sterile medical cotton ball.
  • Item 10 The polypeptide according to any one of items 1-3 or the polypeptide prepared by the method of item 7, the polynucleotide of item 4, the expression vector of item 5, the host cell of item 6, or the composition of item 8 In the preparation of finished products, it is preferably used in medical equipment, tissue engineering products, cosmetics, and health products.
  • the present invention has the following characteristics:
  • the type 17 human collagen sequence selected for the first time in the present invention is a sequence optimized for long-term screening
  • the E. coli expression system is adopted, which is suitable for large-scale amplification. One round of fermentation can be completed in 20 hours, and the production cost is very low. Due to the codon optimization of E. coli and the selection of 2 ⁇ YT medium, The output is very large;
  • the recombinant human collagen produced has very good hydrophilicity and stability, and its amino acid composition is 100% identical to the corresponding part of the amino acid sequence of natural collagen. It will not produce immune rejection and allergic reactions when applied to the human body, and can be widely used. Used in biomedicine and cosmetics industry;
  • the product of the present invention has undergone activity testing and has a biological activity that reaches or exceeds the biological activity of the natural protein of the human body, can exercise the function of the natural protein in the human body, and achieve the purpose of real product application;
  • the technical design of the present invention can effectively reduce the risk of coagulation of collagen when used in the human body, while retaining the high cell adhesion activity of collagen, and has a wide range of tissue engineering application prospects.
  • Figure 1 is a plasmid map constructed by the vectors pET32a-C17A3, pET32a-C17B3, and PET32a-C17C1 of the present invention.
  • Fig. 2 is a protein electrophoresis diagram obtained after expression and purification of the Trx-C17A3 protein of the present invention; the electrophoresis detection molecular weight of the Trx-C17A3 protein is about 42 kDa.
  • Fig. 3 is a protein electrophoresis diagram obtained after expression and purification of the Trx-C17B3 protein of the present invention; the electrophoresis detection molecular weight of the Trx-C17B3 protein is about 40 kDa.
  • Fig. 4 is a protein electrophoresis diagram obtained after expression and purification of the Trx-C17C1 protein of the present invention; the electrophoresis detection molecular weight of the Trx-C17C1 protein is about 32 kDa.
  • Fig. 5 is an electrophoresis diagram of the target protein C17A3 protein obtained after the expression of Trx-C17A3 protein by restriction digestion of Trx tags and ion exchange purification; the electrophoresis detection molecular weight of C17A3 protein is about 25kDa, corresponding to SEQ ID NO.4 The amino acid sequence of the protein.
  • Fig. 6 is an electrophoresis diagram of the target protein C17B3 protein obtained after the expression of Trx-C17B3 protein by restriction digestion of Trx tags and ion exchange purification; the electrophoresis detection molecular weight of C17B3 protein is about 23kDa, corresponding to SEQ ID NO.6 The amino acid sequence of the protein.
  • Fig. 7 is an electrophoresis diagram of the target protein C17C1 protein obtained after expression of Trx-C17C1 protein by restriction digestion of Trx tags and ion exchange purification; the electrophoresis detection molecular weight of C17C1 protein is about 16kDa, corresponding to SEQ ID NO.3 The amino acid sequence of the protein.
  • Fig. 8 is a comparison of the biological activity test results of the C17A3 protein of the present invention with the C17A1 protein (SEQ ID No. 1) and human collagen.
  • Fig. 9 is a comparison of the biological activity detection results of the C17B3 protein of the present invention with the C17B1 protein (SEQ ID No. 2) and human collagen.
  • Figure 10 shows the biological activity test results of the C17C1 protein of the present invention compared with human collagen.
  • medical devices refer to instruments, equipment, appliances, in vitro diagnostic reagents and calibrators, materials, and other similar or related items used directly or indirectly on the human body.
  • tissue engineering product refers to a product used for tissue engineering.
  • Tissue engineering is an emerging discipline that combines cell biology and material science to construct tissues or organs in vitro or in vivo.
  • isolation refers to isolating the target polypeptide from the cultured host cell, for example, to disrupt the host cell and purify the target polypeptide.
  • purification also includes the removal of the Trx or His tag by restriction enzymes.
  • pharmaceutically acceptable carrier is well known to those skilled in the art, and those skilled in the art can select a pharmaceutically acceptable carrier suitable for use in the composition or product of the present invention.
  • pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphoric acid, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl chloride) Ammonium chloride; hexamethylene chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; Tea phenol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; Hydrophil
  • sequence of human type 17 collagen COL17A1 is selected for screening and optimization.
  • the sequence of the human collagen type 17 is the NCBI reference sequence: Q9UMD9.3 (SEQ ID No. 9), see https://www.ncbi.nlm.nih.gov/protein/Q9UMD9.3.
  • the bold underlined part in the above sequence is the amino acid sequence selected in the present invention.
  • the polypeptide is not the full-length sequence of SEQ ID No.9.
  • the present invention is partly based on the discovery that a polypeptide containing at least 63 consecutive amino acid residues in SEQ ID No. 9 can have better biomaterial properties than commercial human collagen, as demonstrated in the examples.
  • Those skilled in the art can appropriately select the consecutive amino acid residues constituting the recombinant collagen.
  • the length of consecutive amino acid residues can be 48-100, 50-72, 54-57, 48-72, and so on.
  • the polypeptide herein can be recombinant human collagen C17A3, which is a triple repeat sequence of C17A, including 207 amino acids, and the basic repeat unit is:
  • GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPG MEGPMGQRGREGPMGPRGEA SEQ ID No. 1
  • amino acid sequence of C17A3 is as follows:
  • the DNA sequence of C17A3 is as follows:
  • the polypeptide herein may be human collagen C17B3, which is a triple repeat sequence of C17B, including 189 amino acids, and the basic repeat unit is:
  • GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA SEQ ID No. 2
  • amino acid sequence of C17B3 is as follows:
  • the DNA sequence of C17B3 is as follows:
  • the polypeptide herein can be human collagen C17C1, which is a repeat of C17C, including 119 amino acids, and the basic repeating unit is:
  • GADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQGPPGQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ (SEQ ID No. 3) is a human collagen type 17 peptide.
  • amino acid sequence of C17C1 is as follows:
  • the DNA sequence of C17C1 is as follows:
  • the polypeptide may include any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID NO: 9
  • One or more, preferably 2, 3, 4, or 5 amino acid residues are substituted, added, deleted or inserted in the amino acid sequence or the same as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3.
  • the amino acid sequence shown in any one of SEQ ID No. 4, SEQ ID No. 6 and SEQ ID NO: 9 has 83%, 84%, 85%, 86%, 87%, 88%, 89%, Amino acid sequences with 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity.
  • the "percent amino acid sequence identity" relative to the reference polypeptide sequence is defined as when the candidate sequence is compared with the reference polypeptide sequence and gaps are introduced when necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered sequence identity After the sexual part, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence.
  • the alignment used to determine the percentage of amino acid sequence identity can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.
  • Amino acid addition refers to the C-terminal of any of the amino acid sequence, such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID NO: 9 Or add amino acids at the N-terminal, as long as the polypeptide has collagen characteristics and cell adhesion activity.
  • Amino acid substitution refers to the amino acid sequence, such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID NO: 9
  • a certain amino acid residue in a certain position is replaced by another amino acid residue, as long as the polypeptide has collagen characteristics and cell adhesion activity.
  • Amino acid insertion refers to the appropriateness of the amino acid sequence, such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID NO: 9.
  • Amino acid residues are inserted at the positions, and the inserted amino acid residues can also be adjacent to each other in whole or in part, or the inserted amino acids are not adjacent to each other, as long as the polypeptide has collagen characteristics and cell adhesion activity.
  • Amino acid deletion refers to the amino acid sequence, such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID NO: 9 1, 2, or more than 3 amino acids are deleted from the above, as long as the polypeptide has collagen characteristics and cell adhesion activity.
  • substitutions may be conservative amino acid substitutions, referring to those in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID NO: 9. Compared with any amino acid sequence, there are 3, more preferably 2 amino acids or 1 amino acid replaced by amino acids with similar or similar properties to form a peptide. These conservative variant peptides can be produced according to Table 1 by performing amino acid substitutions.
  • All amino acids in the polypeptide sequence herein can be L-type amino acids, and one or more (such as 2-5, 2-4 or 2-3) amino acids can also be D-shaped amino acids or artificially modified.
  • D-type amino acids refer to the amino acids that correspond to the L-type amino acids that make up proteins
  • artificially modified amino acids refer to common L-type amino acids that have been modified by methylation, phosphorylation, etc., to make up proteins
  • rare amino acids that exist in nature include those that make up proteins.
  • Uncommon amino acids and amino acids that do not constitute protein such as 5-hydroxylysine, methylhistidine, gamma aminobutyric acid, homoserine, etc.
  • recombinant human collagen can be carried out by conventional methods in the art. For example, it can be produced in the following steps: (1) construction of E. coli genetically engineered bacteria; (2) fermentation and culture of E. coli genetically engineered bacteria; (3) induction and expression of recombinant human collagen; and (4) recombinant human origin Purification of collagen and optional digestion.
  • step (1) the construction of E. coli genetically engineered bacteria can be carried out as follows: (1) Codon optimization and splicing recombination of the DNA fragments in the gene helix region of human-derived type 17 collagen are performed by PCR, and finally the goal is obtained. Gene fragment; (2) Insert the obtained target gene fragment into the PET-32a expression vector to obtain a recombinant expression plasmid; (3) Transfer the recombinant expression plasmid into E. coli competent cells BL21 (DE3), and screen to obtain positive E. coli genes Engineering bacteria.
  • the fermentation culture of E. coli genetically engineered bacteria and the induction and expression of recombinant human collagen can be carried out as follows: (1) Select the optimized E. coli genetically engineered bacteria from the LAB plate A single colony is placed in 10ml of LB medium at 37°C and 220 rpm for 12-16 hours; (2) The bacterial solution is inoculated into 2 ⁇ YT medium at a ratio of 1:100, and then cultured at 37°C for about 3 hours. When the OD 600 was 0.4-0.6, the final concentration of 0.5mM IPTG was added for induction, and the culture was continued at 16°C for 20 hours, and the cells were collected by centrifugation.
  • step (4) the purification and enzyme digestion of recombinant human collagen polypeptide can be carried out as follows: (1) Resuspend bacteria in phosphate buffer (40mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8), and ultrasonically disrupt. Collect the supernatant by centrifugation; (2) Use NI-NTA affinity column to bind recombinant human collagen, 10mM imidazole to rinse the protein, add Tev protease (Tobacco Etch Virus enzyme) at 4°C, digest on the column for 16 hours, and finally Target collagen peptide.
  • phosphate buffer 40mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8
  • Tev protease Tobacco Etch Virus enzyme
  • the host cell may be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Enterobacteriaceae, such as Escherichia coli. It should be understood that those skilled in the art can replace the aforementioned E. coli strains with other expression strains as host cells.
  • Example 1 Construction, expression and purification of recombinant human collagen polypeptide
  • the C17A3 gene has a full length of 621bp.
  • the optimized C17A3 codon gene sequence SEQ ID No. 5, Beijing Shengyuan Kemeng Gene Biotechnology Co., Ltd. is entrusted to synthesize the gene fragment and connect the synthesized C17A3 gene fragment After the Tev protease cleavage site, insert the PET32a expression vector (provided by the Institute of Biophysics, Chinese Academy of Sciences) through the Kpn I and Xho I cleavage sites.
  • the successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3) (Merck). The specific process is as follows: 1: Take 1 ⁇ l of the plasmid in 100 ⁇ l of E.
  • coli competent cells BL21 (DE3), and let stand on ice for 30 minutes.
  • 2 Heat the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes.
  • 3 Add 600 ⁇ l of non-resistant LB to the mixture and incubate for 1 hour at 37° C. and 220 rpm.
  • 4 Take 200 ⁇ l of the bacterial solution and evenly spread it on the LB plate containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 ⁇ g/ml ampicillin antibiotic).
  • 5 Incubate the plate upside down in a 37°C incubator, and cultivate for about 20 hours until clear and visible colonies grow.
  • phosphate buffer pH 7.8 (40mM sodium dihydrogen phosphate, 500mM sodium chloride) to resuspend (1L) the bacterial pellet in about 50ml, and use a high-pressure sterilizer (Xinzhi Bio) to break the bacteria, 13000rpm Centrifuge for 30 minutes to fully separate the soluble protein from the inclusion bodies.
  • Trx-tagged target protein you can directly use an elution buffer (250 mM imidazole, 40 mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8) to elute the target protein Trx-C17A3. If you need to remove the Trx-tagged target protein, you can add an appropriate amount of TEV protease with His tag. After incubating at 4°C for 16 hours, collect the flow-through fluid, which is the target collagen C17A3 from which the carrier protein Trx is removed.
  • elution buffer 250 mM imidazole, 40 mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8
  • TEV protease with His tag you can add an appropriate amount of TEV protease with His tag. After incubating at 4°C for 16 hours, collect the flow-through fluid, which is the target collagen C17A3 from which the carrier protein Trx is removed.
  • the target protein was dialyzed into buffer A (20mM Tris, 15mM NaCl, pH 8.0), flowed through the anion exchange column Hitrap Q (GE Healthcare), and eluted with a gradient of buffer B (20mM Tris, 1M NaCl, pH 8.0), Collect different elution fractions to detect protein.
  • the obtained target protein product was dialyzed overnight, and lyophilized into a dry powder for later use.
  • the molecular weight and purity of the obtained C17A3 protein were detected by SDS-PAGE.
  • the specific process is: take 40 ⁇ l of purified protein solution, add 10 ⁇ l 5 ⁇ protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% ⁇ -mercaptoethanol), boil in boiling water at 100°C for 10 minutes, then add 10 ⁇ l per well to SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue Staining Solution (0.1% Coomassie Brilliant Blue R-250, 25 % Isopropanol, 10% glacial acetic acid) for protein staining for 20 minutes, and then using protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. Finally, the protein activity was measured in comparison with human natural collagen.
  • the C17B3 gene is 567bp in length. According to the optimized C17B3 codon gene sequence SEQ ID No.7, Beijing Shengyuan Kemeng Gene Biotechnology Co., Ltd. is entrusted to synthesize the gene fragment and connect the synthesized C17B3 gene fragment After the Tev protease cleavage site, insert the PET32a expression vector (provided by the Institute of Biophysics, Chinese Academy of Sciences) through the Kpn I and Xho I cleavage sites. The successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3) (Merck). The specific process is as follows: 1: Take 1 ⁇ l of the plasmid in 100 ⁇ l of E.
  • coli competent cells BL21 (DE3), and let stand on ice for 30 minutes.
  • 2 Heat the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes.
  • 3 Add 600 ⁇ l of non-resistant LB to the mixture and incubate for 1 hour at 37° C. and 220 rpm.
  • 4 Take 200 ⁇ l of the bacterial solution and evenly spread it on the LB plate containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 ⁇ g/ml ampicillin antibiotic).
  • 5 Incubate the plate upside down in a 37°C incubator, and cultivate for about 20 hours until clear and visible colonies grow.
  • phosphate buffer pH 7.8 (40mM sodium dihydrogen phosphate, 500mM sodium chloride) to resuspend (1L) the bacterial pellet in about 50ml, and use a high-pressure sterilizer (Xinzhi Bio) to break the bacteria, 13000rpm Centrifuge for 30 minutes to fully separate the soluble protein from the inclusion bodies.
  • Trx-tagged target protein you can directly use an elution buffer (250mM imidazole, 40mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8) to elute the target protein Trx-C17B3. If you need to remove the Trx-tagged target protein, you can add an appropriate amount of TEV protease with His tag. After incubating at 4°C for 16h, collect the flow-through fluid, which is the target collagen C17B3 from which the carrier protein Trx is removed.
  • elution buffer 250mM imidazole, 40mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8
  • TEV protease with His tag After incubating at 4°C for 16h, collect the flow-through fluid, which is the target collagen C17B3 from which the carrier protein Trx is removed.
  • the target protein was dialyzed into buffer A (20mM Tris, 15mM NaCl, pH 8.0), flowed through the anion exchange column Hitrap Q (GE Healthcare), and eluted with a gradient of buffer B (20mM Tris, 1M NaCl, pH 8.0), Collect different elution fractions to detect protein.
  • the obtained target protein product was dialyzed overnight, and lyophilized into a dry powder for later use.
  • the molecular weight and purity of the obtained C17B3 protein were detected by SDS-PAGE.
  • the specific process is: take 40 ⁇ l of purified protein solution, add 10 ⁇ l 5 ⁇ protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% ⁇ -mercaptoethanol), boil in boiling water at 100°C for 10 minutes, then add 10 ⁇ l per well to SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue Staining Solution (0.1% Coomassie Brilliant Blue R-250, 25 % Isopropanol, 10% glacial acetic acid) for protein staining for 20 minutes, and then using protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. Finally, the protein activity was measured in comparison with human natural collagen.
  • the C17C1 gene is 357bp in length.
  • the optimized C17C1 codon gene sequence SEQ ID No. 8, Beijing Shengyuan Kemeng Gene Biotechnology Co., Ltd. is entrusted to synthesize the gene fragment, and the synthesized C17C1 gene fragment is connected After the Tev protease cleavage site, insert the PET32a expression vector (provided by the Institute of Biophysics, Chinese Academy of Sciences) through the Kpn I and Xho I cleavage sites.
  • the successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3) (Merck). The specific process is as follows: 1: Take 1 ⁇ l of the plasmid in 100 ⁇ l of E.
  • coli competent cells BL21 (DE3), and let stand on ice for 30 minutes.
  • 2 Heat the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes.
  • 3 Add 600 ⁇ l of non-resistant LB to the mixture and incubate for 1 hour at 37° C. and 220 rpm.
  • 4 Take 200 ⁇ l of the bacterial solution and evenly spread it on the LB plate containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 ⁇ g/ml ampicillin antibiotic).
  • 5 Incubate the plate upside down in a 37°C incubator, and cultivate for about 20 hours until clear and visible colonies grow.
  • phosphate buffer pH 7.8 (40mM sodium dihydrogen phosphate, 500mM sodium chloride) to resuspend (1L) the bacterial pellet in about 50ml, and use a high-pressure sterilizer (Xinzhi Bio) to break the bacteria, 13000rpm Centrifuge for 30 minutes to fully separate the soluble protein from the inclusion bodies.
  • Trx-tagged target protein you can directly use an elution buffer (250mM imidazole, 40mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8) to elute the target protein Trx-C17C1. If you need to remove the Trx-tagged target protein, you can add an appropriate amount of TEV protease with His tag. After incubating at 4°C for 16 hours, collect the flow-through fluid, which is the target collagen C17C1 from which the carrier protein Trx is removed.
  • elution buffer 250mM imidazole, 40mM NaH 2 PO 3 , 500 mM NaCl, pH 7.8
  • TEV protease with His tag you can add an appropriate amount of TEV protease with His tag. After incubating at 4°C for 16 hours, collect the flow-through fluid, which is the target collagen C17C1 from which the carrier protein Trx is removed.
  • the target protein was dialyzed into buffer A (20mM Tris, 15mM NaCl, pH 8.0), flowed through the anion exchange column Hitrap Q (GE Healthcare), and eluted with a gradient of buffer B (20mM Tris, 1M NaCl, pH 8.0), Collect different elution fractions to detect protein.
  • the obtained target protein product was dialyzed overnight, and lyophilized into a dry powder for later use.
  • the molecular weight and purity of the obtained C17C1 protein were detected by SDS-PAGE.
  • the specific process is: take 40 ⁇ l of purified protein solution, add 10 ⁇ l 5 ⁇ protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% ⁇ -mercaptoethanol), boil in boiling water at 100°C for 10 minutes, then add 10 ⁇ l per well to SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue Staining Solution (0.1% Coomassie Brilliant Blue R-250, 25 % Isopropanol, 10% glacial acetic acid) for protein staining for 20 minutes, and then using protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. Finally, the protein activity was measured in comparison with human natural collagen.
  • Figures 2 to 4 show the electrophoresis diagrams of Trx-C17A3, Trx-C17B3 and Trx-C17C1 fusion proteins with apparent molecular weights of 42kDa, 40kDa and 32kDa, respectively.
  • the principle of this method is to determine the characteristic absorption of peptide bonds under far ultraviolet light, which is not affected by the content of chromophore, has less interference substances, and is simple to operate. It is suitable for detecting human collagen and its analogs that are not colored by Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After detecting the protein concentration, adjust the concentration of all tested proteins to 0.5 mg/ml with PBS.
  • each well was added 10 5 good state cultured 3T3 cells (Pei children from Tsinghua University teachers) and incubated 60min 37 °C.
  • the absorbance at OD 492nm can reflect the cell adhesion activity of collagen or its fragments. The higher the cell adhesion activity of the protein, the more it can provide cells with a high-quality external environment in a short time and help cells adhere to the wall.
  • Figs. 8-10 are plotted based on the average and standard error of the OD 492nm of three parallel experiments.

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Abstract

本发明提供了一种多肽，所述多肽包含SEQ ID No.9的63至1496个连续的氨基酸残基，其中所述多肽包含(A) m所示的序列或者由(A) m所示的序列组成，其中每个A为选自SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个所示的氨基酸序列或SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个取代、添加、或缺失了1或多个，例如2、3、4或5个氨基酸残基的氨基酸序列或者与SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个所示的氨基酸序列具有83％-97％序列同一性的序列；m为1-10之间的整数；其中每个A相同或不同并且相邻两个A之间直接通过肽键连接或者通过1个以上的氨基酸残基连接；其中所述多肽具有细胞粘附活性，以及所述多肽的生产方法和用途。

Description

人胶原蛋白17型多肽、其生产方法和用途
本申请要求于2019年10月31日递交的中国专利申请第201911051106.3号的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本发明属于基因工程技术领域，涉及多肽、其生产方法和用途。
背景技术
胶原蛋白
胶原蛋白一般为白色、透明、无分支的原纤维，是皮肤和骨骼的基础支撑物，可以占到蛋白质总量的25％～35％，主要分布于人体的皮肤、血管、骨骼、筋腱、牙齿和软骨等处，是这些组织的主要基质和支架，保护并连结各种组织，在体内发挥着重要的生理功能。因此，胶原蛋白可以广泛的应用在医药和化妆品等行业中。
当前市场上销售的胶原蛋白产品都是取自猪、牛、鱼等动物组织中。虽然某些动物与人的胶原蛋白相似度较高，其仍然难以避免病毒感染以及致敏性的危险。目前少部分动物来源的胶原蛋白已经运用于化妆品中，但很难被广泛用于医疗器材或较精密的组织工程产品中，根本无法发挥胶原蛋白的原本生物学功能。而且，常规方法制备的胶原蛋白一般具有较强的凝血功能，导致其应用于某些组织工程产品中的时候，带来很大的血栓形成的风险，极大地限制了其广泛深入的应用。
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本身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。国内外一些研究机构通过常规重组表达的方法体外表达人源胶原蛋白，但是通常生产的成本过高，生产周期过长，无法投入大规模生产。因此市场上急需一种具有优良生物材料性质，氨基酸序列与人体高度同源，且能在工业化体系中大量制备的胶原蛋白材料。
17型人胶原蛋白
从结构上来说，人体天然的胶原蛋白的结构非常的复杂，所以才导致人源胶原蛋白极难通过常规手段表达和大量制备。胶原蛋白最普遍的结构特征是由3条肽链形成的三螺旋结构，即由3条A肽链以右手超螺旋方式形成蛋白质，这样的三股螺旋区域被称为胶原区域。每个A肽链在分子结构上都是由重复出现的Gly-X-Y(X、Y代表Gly之外的任何氨基酸残基，X往往是Pro，Y往往是Hyp)肽段构成左手螺旋，3条链在氨基酸残基的相互作用下，以同一轴为中心，以右手超螺旋方式形成稳定的三股螺旋结构。因此，一般胶原蛋白的序列很难自发结合成为稳定的三螺旋结构发挥生物学功能，这样的困难严重阻碍了人胶原蛋白的研发和生产。
人体中含有28种不同型别的胶原蛋白，分为常见的纤维类胶原蛋白和不常见的非纤维类胶原蛋白。人体皮肤中I型，II型，III型等都属于纤维类胶原蛋白。在非纤维类胶原蛋白中，有一个非常重要的胶原蛋白亚型就是17型胶原蛋白collagen XVII(人体中由COL17A1基因编码)。17型胶原蛋白是由三个COL17A1链结合形成的同源三聚体，单链分子量180kDa。其中包含有70kDa的球状胞内结构域，一个跨膜域和120kDa的胞外胶原结构域，具有非常强的热稳定性。近期研究证实，17型胶原蛋白在人体中是表皮干细胞半桥粒的重要组成成分，对于细胞衰老和皮肤分化都具有重要的作用。然而，目前人类对于非纤维类胶原蛋白的结构功能认识非常有限，尤其是对于17型胶原蛋白知之甚少。
发明人对于胶原蛋白的结构和功能已经深入研究多年，特别是国际首次解析了人胶原蛋白多个区段的全新的原子结构，并投递到国际蛋白质结构数据库中公开展示，积累了丰富的研究经验。通过反复摸索，发明人已经成功实现了17型胶原蛋白胞外若干功能区的重组表达，发现其具有优良的生物材料特性，其制备方法简单，易于扩大生产，可以广泛的应用在医药和化妆品等行业中。
发明内容
本发明部分基于以下发现：
本发明的多肽C17A3、C17B3和C17C1与现有人胶原蛋白相比具有相当或更高的细胞粘附效果，并且多肽C17A3、C17B3和C17C1在宿主 细胞中表达后以水溶形式存在，且制备方法简单，易于扩大生产。
针对背景技术所示的现有技术缺陷，本发明提供了：
项目1.多肽，所述多肽包含SEQ ID No.9中的63至1496个连续的氨基酸残基，其中所述多肽具有细胞粘附活性。
项目2.多肽，其中所述多肽包含(A) m所示的序列或者由(A) m所示的序列组成，其中每个A为选自SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个所示的氨基酸序列或SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个取代、添加、或缺失了1或多个，例如2、3、4或5个氨基酸残基的氨基酸序列或者与SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个所示的氨基酸序列具有83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％或97％序列同一性的序列；m为1-10之间的整数；其中每个A相同或不同并且相邻两个A之间直接通过肽键连接或者通过1个以上的氨基酸残基连接；其中所述多肽具有细胞粘附活性。本文所述的区间包括端点，例如1-10之间包括1、2、3、4、5、6、7、8、9、10，即，m可以为1、2、3、4、5、6、7、8、9或10。
项目3.项目1或2所述的多肽，其中所述多肽包含SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.6所示的氨基酸序列或由其组成。
项目4.多核苷酸，其编码根据项目1-3中任一项所述的多肽，优选地，所述多核苷酸包含SEQ ID No.5、SEQ ID No.7、或SEQ ID No.8所示的核苷酸序列或由SEQ ID No.5、SEQ ID No.7、或SEQ ID No.8所示的核苷酸序列组成。
项目5.表达载体，其包含根据项目4所述的多核苷酸。
项目6.宿主细胞，其包含根据项目5所述的表达载体或者表达项目1-3中任一项所述的多肽，其中所述宿主细胞优选是大肠杆菌细胞。
项目7.制备根据项目1-3中任一项所述的多肽的方法，其包括：
(1)在生产培养基中培养根据项目6所述的宿主细胞；
(2)从宿主细胞分离出项目1-3中任一项所述的多肽。
项目8.组合物，其包含根据项目1-3中任一项所述的多肽或项目7的方法制备的多肽。
项目9.制品，其包含根据项目1-3中任一项所述的多肽或项目7的方 法制备的多肽或者项目8所述的组合物，其中所述制成品为药物组合物、医疗器材、组织工程产品、化妆品或保健品，优选地所述药物组合物是外用制剂，优选外用涂抹制剂，例如外用凝胶剂或外用浸润制剂；其中优选地所述外用凝胶剂还包含药学上可接受的载体，所述外用浸润制剂还包含无菌医用棉球。
项目10.根据项目1-3中任一项所述的多肽或项目7的方法制备的多肽、项目4的多核苷酸、项目5的表达载体、项目6的宿主细胞、或项目8的组合物在制备制成品，优选医疗器材、组织工程产品、化妆品、保健品中的用途。
与现有技术相比，本发明具有以下特点：
(1)本发明首次选择的17型人胶原蛋白序列为长期筛选优化的序列；
(2)采用大肠杆菌表达系统，适于大规模放大，20小时即可完成一轮发酵，生产成本非常低，由于对基因序列进行了大肠杆菌的密码子优化以及选用2×YT培养基，使得产量非常大；
(3)生产的重组人源胶原蛋白具有非常好的亲水性和稳定性，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，可以广泛应用于生物医药和化妆品行业；
(4)本发明产品经过活性检测，具有达到甚至超过人体天然蛋白的生物学活性，可以在人体中行使天然蛋白的功能，达到真正的产品应用的目的；
(5)本发明的技术设计可以有效减少胶原蛋白在人体使用时的凝血风险，同时保留胶原蛋白高细胞粘附活性，具有广泛的组织工程应用前景。
附图说明
图1为本发明载体pET32a-C17A3、pET32a-C17B3、PET32a-C17C1构建的质粒图谱。
图2为本发明Trx-C17A3蛋白表达纯化后得到的蛋白电泳图；Trx-C17A3蛋白的电泳检测分子量约为42kDa。
图3为本发明Trx-C17B3蛋白表达纯化后得到的蛋白电泳图；Trx-C17B3蛋白的电泳检测分子量约为40kDa。
图4为本发明Trx-C17C1蛋白表达纯化后得到的蛋白电泳图；Trx-C17C1蛋白的电泳检测分子量约为32kDa。
图5为本发明Trx-C17A3蛋白表达后经过酶切切除Trx标签和离子交换纯化得到的目的蛋白C17A3蛋白的电泳图；C17A3蛋白的电泳检测分子量约为25kDa，对应于具有SEQ ID NO.4的氨基酸序列的蛋白质。
图6为本发明Trx-C17B3蛋白表达后经过酶切切除Trx标签和离子交换纯化得到的目的蛋白C17B3蛋白的电泳图；C17B3蛋白的电泳检测分子量约为23kDa，对应于具有SEQ ID NO.6的氨基酸序列的蛋白质。
图7为本发明Trx-C17C1蛋白表达后经过酶切切除Trx标签和离子交换纯化得到的目的蛋白C17C1蛋白的电泳图；C17C1蛋白的电泳检测分子量约为16kDa，对应于具有SEQ ID NO.3的氨基酸序列的蛋白质。
图8为本发明C17A3蛋白与C17A1蛋白(SEQ ID No.1)、人胶原蛋白相比较的生物活性检测结果。
图9为本发明C17B3蛋白与C17B1蛋白(SEQ ID No.2)、人胶原蛋白相比较的生物活性检测结果。
图10为本发明C17C1蛋白与人胶原蛋白相比较的生物活性检测结果。
具体实施方式
下文提供进一步的描述以便于理解本发明。
如本文中使用，“医疗器械”是指直接或者间接用于人体的仪器、设备、器具、体外诊断试剂及校准物、材料以及其他类似或者相关的物品。
如本文中使用，“组织工程产品”是指用于组织工程的产品。组织工程是一门以细胞生物学和材料科学相结合，进行体外或体内构建组织或器官的新兴学科。
如本文中使用，“分离”指从培养的宿主细胞中分离出目标多肽，例如，对宿主细胞进行破胞，并纯化出目标多肽。在纯化出的目标多肽带有纯化标签，如Trx或His标签的情况下，“分离”还包括酶切切除Trx或His标签。
“药学上可接受的载体”是本领域技术人员公知的，并且本领域技术人员可以选择适用于本发明组合物或制品中药学上可接受的载体。例如，药学上可接受的载体包括但不限于:缓冲剂如磷酸、柠檬酸和其它有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如十八烷基二甲基苄基氯化铵；氯己双 铵；苯扎氯铵；苄索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(低于约10个残基)多肽；蛋白质，如血清清蛋白、明胶、或免疫球蛋白；亲水性聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖、和其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂如EDTA；糖如蔗糖、甘露醇、海藻糖或山梨糖醇；形成盐的反荷离子如钠；金属复合物(例如Zn-蛋白复合物)；和/或非离子型表面活性剂如聚乙二醇(PEG)。
在本发明中，选择人17型胶原蛋白COL17A1序列进行筛选优化。所述人胶原17型的序列是NCBI参照序列：Q9UMD9.3(SEQ ID No.9)，参见https://www.ncbi.nlm.nih.gov/protein/Q9UMD9.3。
Figure PCTCN2020123600-appb-000001
Figure PCTCN2020123600-appb-000002
上述序列中粗体下划线部分即为本发明选择的氨基酸序列。申请人经过大量的研究发现，选择的上述序列水溶性强，重组表达产量高，纯化工艺简单，且比商品化的人胶原蛋白或SEQ ID No.9中的其他序列实现更好的细胞粘附效果，具有多种优良的生物材料特性。在本发明中，多肽不是SEQ ID No.9的全长序列。
本发明部分基于以下发现：包含SEQ ID No.9中的至少63个连续的氨基酸残基的多肽能够比商品化的人胶原蛋白具有更好的生物材料特性，如实施例证明。本领域技术人员可以适当选择构成重组胶原蛋白的连续的氨基酸残基。例如，连续的氨基酸残基的长度可以是48-100、50-72、54-57、48-72等等。
在本发明中，对几个具体的氨基酸区域的序列进行了测试：
Figure PCTCN2020123600-appb-000003
多肽在本文中可以是重组人源胶原蛋白C17A3，为C17A的三次重复序列，包括氨基酸207个，基本重复单元为：
GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPG MEGPMGQRGREGPMGPRGEA(SEQ ID No.1)，为人胶原蛋白17型肽段。
C17A3的氨基酸序列如下：
Figure PCTCN2020123600-appb-000004
C17A3的DNA序列如下：
Figure PCTCN2020123600-appb-000005
多肽在本文中可以是人源胶原蛋白C17B3，为C17B的三次重复序列，包括189个氨基酸，基本重复单元为：
GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA(SEQ ID No.2)，为人胶原蛋白17型肽段。
C17B3的氨基酸序列如下：
Figure PCTCN2020123600-appb-000006
Figure PCTCN2020123600-appb-000007
C17B3的DNA序列如下：
Figure PCTCN2020123600-appb-000008
多肽在本文中可以是人源胶原蛋白C17C1，为C17C的1次重复，包括氨基酸119个，基本重复单元为：
GADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQGPPGQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ(SEQ ID No.3)，为人胶原蛋白17型肽段。
C17C1的氨基酸序列如下：
Figure PCTCN2020123600-appb-000009
C17C1的DNA序列如下：
Figure PCTCN2020123600-appb-000010
Figure PCTCN2020123600-appb-000011
在本文中，多肽可以包括以SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种所示的氨基酸序列中取代、添加、缺失或插入了1个或多个，优选2、3、4或5个氨基酸残基的氨基酸序列或与SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种所示的氨基酸序列具有83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％或97％序列同一性的氨基酸序列。相对于参考多肽序列的“氨基酸序列同一性百分数”定义为在将候选序列与参考多肽序列进行比对并在必要时引入空位以获取最大百分比序列同一性，且不将任何保守置换视为序列同一性的一部分之后，候选序列中与参考多肽序列中的氨基酸残基相同的氨基酸残基的百分数。用于确定氨基酸序列同一性百分数的比对可以用本领域技术人员熟知的各种方式实现，例如，使用公众可获得的计算机软件，如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的合适参数，包括在所比较的序列的全长上实现最大比对所需的任何算法。
氨基酸添加指在氨基酸序列，例如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种的C端或N端添加氨基酸，只要所述多肽具有胶原蛋白特征和细胞粘附活性。
氨基酸取代指在氨基酸序列，例如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种的序列的某个位置的某个氨基酸残基被其他氨基酸残基替代，只要所述多肽具有胶原蛋白特征和细胞粘附活性。
氨基酸插入指在氨基酸序列例如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种的序列的适当位置插入氨基酸残基，插入的氨基酸残基也可以全部或部分彼此相邻， 或插入的氨基酸之间都不彼此相邻，只要所述多肽具有胶原蛋白特征和细胞粘附活性。
氨基酸缺失指可以从氨基酸序列，例如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种的序列中删除1、2或3个以上氨基酸，只要所述多肽具有胶原蛋白特征和细胞粘附活性。
在本发明中，取代可以是保守氨基酸取代，指与SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.6和SEQ ID NO:9中任一种的氨基酸序列相比，有3个，更佳地2个氨基酸或1个氨基酸被性质相似或相近的氨基酸所替换而形成肽。这些保守性变异肽可以根据表1进行氨基酸替换而产生。
表1：氨基酸保守取代
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本文的多肽序列中的所有氨基酸可为L型氨基酸，其中的一个或多个(如2-5个、2-4个或2-3个)氨基酸也可以用构象为D型的氨基酸、人工 修饰的氨基酸、自然界存在的稀有氨基酸等进行替换，以提高多肽的生物利用度、稳定性和/或抗病毒活性。其中D型氨基酸是指与组成蛋白质的L型氨基酸相对应的氨基酸；人工修饰的氨基酸指经过甲基化、磷酸化等修饰的组成蛋白质的常见L型氨基酸；自然界存在的稀有氨基酸包括组成蛋白质的不常见氨基酸和不组成蛋白质的氨基酸，例如5-羟基赖氨酸、甲基组氨酸、γ氨基丁酸、高丝氨酸等。
在本发明中，重组人源胶原蛋白可以通过本领域中常规的方法进行。例如，可以如下步骤生产：(1)大肠杆菌基因工程菌的构建；(2)大肠杆菌基因工程菌的发酵培养；(3)重组人源胶原蛋白的诱导和表达；以及(4)重组人源胶原蛋白的纯化和任选的酶切。
在步骤(1)中，大肠杆菌基因工程菌的构建可以如下进行：(1)利用PCR方法对人源性17型胶原蛋白的基因螺旋区的DNA片段进行密码子优化和拼接重组，最终得到目的基因片段；(2)将得到的目的基因片段插入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。
在步骤(2)与(3)中，大肠杆菌基因工程菌的发酵培养和重组人源胶原蛋白的诱导和表达可以如下进行：(1)从LAB平板中挑取优选后的大肠杆菌基因工程菌单菌落，置于10ml的LB培养基中37℃，220rpm培养12-16小时；(2)将菌液按照1：100接种到2×YT培养基中放大培养，37℃培养约3小时，待OD 600在0.4-0.6时，加入终浓度为0.5mM IPTG进行诱导，16℃继续培养20小时，离心收集菌体。
在步骤(4)中，重组人源胶原蛋白多肽的纯化和酶切可以如下进行：(1)用磷酸盐缓冲液(40mM NaH 2PO 3，500mM NaCl，pH 7.8)重悬细菌，超声破碎，离心收集上清液；(2)利用NI-NTA亲和柱结合重组人源胶原蛋白，10mM咪唑漂洗杂蛋白后，加入Tev蛋白酶(Tobacco Etch Virus enzyme)4℃，16h柱上酶切，最后获得目的胶原蛋白多肽。
宿主细胞可以是真核细胞，例如真菌和酵母，原核细胞，例如肠杆菌科细菌，如大肠杆菌。应当理解，本领域技术人员可以通过用其它表达菌株替换上述大肠杆菌菌株作为宿主细胞。
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：重组人源胶原蛋白多肽的构建、表达与纯化
C17A3基因表达载体的构建及表达
1.实施例1中使用的人源胶原蛋白C17A3全长基因序列以SEQ ID No.5显示。该序列已经针对大肠杆菌的密码子进行了密码子优化。
2.C17A3基因全长621bp，根据优化后的C17A3密码子基因序列SEQ ID No.5，委托北京盛元科萌基因生物科技有限公司进行基因片段的合成，并将合成后的C17A3基因片段连接上Tev蛋白酶切位点之后通过Kpn I和Xho I的酶切位点插入PET32a表达载体(由中科院生物物理所提供)。将该构建成功的表达质粒转化大肠杆菌感受态细胞BL21(DE3)(Merck公司)。具体过程为：1：取1μl的该质粒于100μl的大肠杆菌感受态细胞BL21(DE3)中，冰上静置30min。2：将该混合物于42℃水浴锅中热激90s，然后迅速置于冰上静置2min。3：向该混合物中加入600μl无抗性的LB，37℃，220rpm条件下培养1h。4：取200μl该菌液均匀的涂布在含有氨苄青霉素抗性的LB平板上(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠，15g/L琼脂，100μg/ml氨苄抗生素)。5：将平板倒置培养于37℃温箱中，培养约20h待长出清晰可见的菌落。
3.从转化好的LB平板中挑取单克隆菌落于10ml LB(含100μg/ml氨苄抗生素)培养基中培养12h-16h后，再按照1:100的比例转接到2×YT培养基(16g/L蛋白胨，10g/L酵母提取物，5g/L氯化钠)中进行扩大培养，37℃，220rpm培养至菌液OD600在0.4-0.6时，加入终浓度为0.5mM IPTG(Sigma公司，货号：I5502-1G)进行诱导表达，诱导条件为18℃、180rpm培养20h。最后离心收集菌体，保存于-20℃或者立即进入下步纯化。
4.用磷酸盐缓冲液(pH 7.8)(40mM磷酸二氢钠，500mM氯化钠)约50ml重悬(1L)菌体沉淀，利用高压破菌仪器(新芝生物)进行破菌后，13000rpm离心30min，使可溶性蛋白与包涵体充分分离。
5.用5倍柱体积的结合缓冲液(Binding buffer)(40mM NaH 2PO 3，500mM NaCl，pH 7.8)平衡Ni-NTA(Qiagen公司，货号：30210)亲和柱。然后加入蛋白上清于4℃条件下孵育0.5-1h，使目的重组蛋白充分结合到柱 材上。再用200ml含有10mM咪唑(Sigma公司)的洗涤缓冲液(washing buffer)(10mM咪唑，40mM NaH 2PO 3，500mM NaCl，pH 7.8)漂洗杂蛋白。如果需要带Trx标签的目的蛋白，可以直接用洗脱缓冲液(elution buffer)(250mM咪唑，40mM NaH 2PO 3，500mM NaCl，pH 7.8)洗脱目的蛋白Trx-C17A3。如果需要切除Trx标签的目的蛋白，可以加入适量具有His标签的TEV蛋白酶，于4℃孵育16h后，收集流穿液，即为去除载体蛋白Trx的目的胶原蛋白C17A3。
6.用阴离子交换柱可以进行目的蛋白快速纯化。将目的蛋白透析到缓冲液A(20mM Tris，15mM NaCl，pH 8.0)中，流穿阴离子交换柱Hitrap Q(GE Healthcare)，并用缓冲液B(20mM Tris，1M NaCl，pH 8.0)梯度洗脱，收集不同洗脱组分检测蛋白。所得目的蛋白产物透析过夜，冻干为干粉待用。
7.所得C17A3蛋白利用SDS-PAGE检测分子量和纯度。具体过程为：取纯化后的蛋白液40μl，加入10μl 5×的蛋白上样缓冲液(250mM的Tris-HCl(pH:6.8)，10％SDS,0.5％溴酚蓝，50％甘油，5％β-巯基乙醇)，置于100℃沸水中煮10min，然后每孔10μl加入SDS-PAGE蛋白胶中，电压80V跑2h后，用考马斯亮蓝染色液(0.1％考马斯亮蓝R-250，25％异丙醇，10％冰醋酸)进行蛋白染色20min，再利用蛋白脱色液(10％醋酸，5％乙醇)进行脱色。最后与人天然胶原蛋白相对照测量蛋白活性。
C17B3基因表达载体的构建及表达
1.实施例2中使用的人源胶原蛋白C17B3全长基因序列以SEQ ID No.7显示。该序列已经针对大肠杆菌的密码子进行了密码子优化。
2.C17B3基因全长567bp，根据优化后的C17B3密码子基因序列SEQ ID No.7，委托北京盛元科萌基因生物科技有限公司进行基因片段的合成，并将合成后的C17B3基因片段连接上Tev蛋白酶切位点之后通过Kpn I和Xho I的酶切位点插入PET32a表达载体(由中科院生物物理所提供)。将该构建成功的表达质粒转化大肠杆菌感受态细胞BL21(DE3)(Merck公司)。具体过程为：1：取1μl的该质粒于100μl的大肠杆菌感受态细胞BL21(DE3)中，冰上静置30min。2：将该混合物于42℃水浴锅中热激90s，然后迅速置于冰上静置2min。3：向该混合物中加入600μl无抗性的LB，37℃，220rpm 条件下培养1h。4：取200μl该菌液均匀的涂布在含有氨苄青霉素抗性的LB平板上(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠，15g/L琼脂，100μg/ml氨苄抗生素)。5：将平板倒置培养于37℃温箱中，培养约20h待长出清晰可见的菌落。
3.从转化好的LB平板中挑取单克隆菌落于10ml LB(含100μg/ml氨苄抗生素)培养基中培养12h-16h后，再按照1:100的比例转接到2×YT培养基(16g/L蛋白胨，10g/L酵母提取物，5g/L氯化钠)中进行扩大培养，37℃，220rpm培养至菌液OD600在0.4-0.6时，加入终浓度为0.5mM IPTG(Sigma公司，货号：I5502-1G)进行诱导表达，诱导条件为18℃、180rpm培养20h。最后离心收集菌体，保存于-20℃或者立即进入下步纯化。
4.用磷酸盐缓冲液(pH 7.8)(40mM磷酸二氢钠，500mM氯化钠)约50ml重悬(1L)菌体沉淀，利用高压破菌仪器(新芝生物)进行破菌后，13000rpm离心30min，使可溶性蛋白与包涵体充分分离。
5.用5倍柱体积的结合缓冲液(Binding buffer)(40mM NaH 2PO 3，500mM NaCl，pH 7.8)平衡Ni-NTA(Qiagen公司，货号：30210)亲和柱。然后加入蛋白上清于4℃条件下孵育0.5-1h，使目的重组蛋白充分结合到柱材上。再用200ml含有10mM咪唑(Sigma公司)的洗涤缓冲液(washing buffer)(10mM咪唑，40mM NaH 2PO 3，500mM NaCl，pH 7.8)漂洗杂蛋白。如果需要带Trx标签的目的蛋白，可以直接用洗脱缓冲液(elution buffer)(250mM咪唑，40mM NaH 2PO 3，500mM NaCl，pH 7.8)洗脱目的蛋白Trx-C17B3。如果需要切除Trx标签的目的蛋白，可以加入适量具有His标签的TEV蛋白酶，于4℃孵育16h后，收集流穿液，即为去除载体蛋白Trx的目的胶原蛋白C17B3。
6.用阴离子交换柱可以进行目的蛋白快速纯化。将目的蛋白透析到缓冲液A(20mM Tris，15mM NaCl，pH 8.0)中，流穿阴离子交换柱Hitrap Q(GE Healthcare)，并用缓冲液B(20mM Tris，1M NaCl，pH 8.0)梯度洗脱，收集不同洗脱组分检测蛋白。所得目的蛋白产物透析过夜，冻干为干粉待用。
7.所得C17B3蛋白利用SDS-PAGE检测分子量和纯度。具体过程为：取纯化后的蛋白液40μl，加入10μl 5×的蛋白上样缓冲液(250mM的Tris-HCl(pH:6.8)，10％SDS,0.5％溴酚蓝，50％甘油，5％β-巯基乙醇)，置 于100℃沸水中煮10min，然后每孔10μl加入SDS-PAGE蛋白胶中，电压80V跑2h后，用考马斯亮蓝染色液(0.1％考马斯亮蓝R-250，25％异丙醇，10％冰醋酸)进行蛋白染色20min，再利用蛋白脱色液(10％醋酸，5％乙醇)进行脱色。最后与人天然胶原蛋白相对照测量蛋白活性。
C17C1基因表达载体的构建及表达
1.实施例2中使用的人源胶原蛋白C17C1全长基因序列以SEQ ID No.8显示。该序列已经针对大肠杆菌的密码子进行了密码子优化。
2.C17C1基因全长357bp，根据优化后的C17C1密码子基因序列SEQ ID No.8，委托北京盛元科萌基因生物科技有限公司进行基因片段的合成，并将合成后的C17C1基因片段连接上Tev蛋白酶切位点之后通过Kpn I和Xho I的酶切位点插入PET32a表达载体(由中科院生物物理所提供)。将该构建成功的表达质粒转化大肠杆菌感受态细胞BL21(DE3)(Merck公司)。具体过程为：1：取1μl的该质粒于100μl的大肠杆菌感受态细胞BL21(DE3)中，冰上静置30min。2：将该混合物于42℃水浴锅中热激90s，然后迅速置于冰上静置2min。3：向该混合物中加入600μl无抗性的LB，37℃，220rpm条件下培养1h。4：取200μl该菌液均匀的涂布在含有氨苄青霉素抗性的LB平板上(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠，15g/L琼脂，100μg/ml氨苄抗生素)。5：将平板倒置培养于37℃温箱中，培养约20h待长出清晰可见的菌落。
3.从转化好的LB平板中挑取单克隆菌落于10ml LB(含100μg/ml氨苄抗生素)培养基中培养12h-16h后，再按照1:100的比例转接到2×YT培养基(16g/L蛋白胨，10g/L酵母提取物，5g/L氯化钠)中进行扩大培养，37℃，220rpm培养至菌液OD600在0.4-0.6时，加入终浓度为0.5mM IPTG(Sigma公司，货号：I5502-1G)进行诱导表达，诱导条件为18℃、180rpm培养20h。最后离心收集菌体，保存于-20℃或者立即进入下步纯化。
4.用磷酸盐缓冲液(pH 7.8)(40mM磷酸二氢钠，500mM氯化钠)约50ml重悬(1L)菌体沉淀，利用高压破菌仪器(新芝生物)进行破菌后，13000rpm离心30min，使可溶性蛋白与包涵体充分分离。
5.用5倍柱体积的结合缓冲液(Binding buffer)(40mM NaH 2PO 3，500mM NaCl，pH 7.8)平衡Ni-NTA(Qiagen公司，货号：30210)亲和柱。然 后加入蛋白上清于4℃条件下孵育0.5-1h，使目的重组蛋白充分结合到柱材上。再用200ml含有10mM咪唑(Sigma公司)的洗涤缓冲液(washing buffer)(10mM咪唑，40mM NaH 2PO 3，500mM NaCl，pH 7.8)漂洗杂蛋白。如果需要带Trx标签的目的蛋白，可以直接用洗脱缓冲液(elution buffer)(250mM咪唑，40mM NaH 2PO 3，500mM NaCl，pH 7.8)洗脱目的蛋白Trx-C17C1。如果需要切除Trx标签的目的蛋白，可以加入适量具有His标签的TEV蛋白酶，于4℃孵育16h后，收集流穿液，即为去除载体蛋白Trx的目的胶原蛋白C17C1。
6.用阴离子交换柱可以进行目的蛋白快速纯化。将目的蛋白透析到缓冲液A(20mM Tris，15mM NaCl，pH 8.0)中，流穿阴离子交换柱Hitrap Q(GE Healthcare)，并用缓冲液B(20mM Tris，1M NaCl，pH 8.0)梯度洗脱，收集不同洗脱组分检测蛋白。所得目的蛋白产物透析过夜，冻干为干粉待用。
7.所得C17C1蛋白利用SDS-PAGE检测分子量和纯度。具体过程为：取纯化后的蛋白液40μl，加入10μl 5×的蛋白上样缓冲液(250mM的Tris-HCl(pH:6.8)，10％SDS,0.5％溴酚蓝，50％甘油，5％β-巯基乙醇)，置于100℃沸水中煮10min，然后每孔10μl加入SDS-PAGE蛋白胶中，电压80V跑2h后，用考马斯亮蓝染色液(0.1％考马斯亮蓝R-250，25％异丙醇，10％冰醋酸)进行蛋白染色20min，再利用蛋白脱色液(10％醋酸，5％乙醇)进行脱色。最后与人天然胶原蛋白相对照测量蛋白活性。
结果
图2-图4的电泳图分别表明得到表观分子量42kDa、40kDa和32kDa的Trx-C17A3，Trx-C17B3和Trx-C17C1融合蛋白。
图5-图7的电泳图分别表明得到表观分子量25kDa，23kDa，16kDa的C17A3、C17B3和C17C1融合蛋白。
实施例2 C17A3、C17B3、C17C1蛋白的细胞粘附活性检测
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，包括对照人胶原蛋白(Sigma，C7774)、C17A3、C17A1(SEQ ID No.1，与C17A3相同方法制备)、C17B3、C17B1(SEQ ID No.2，与C17B3相同方法制备)、C17C1蛋白样品。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)＝144X(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.Humana Press.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10 5个培养状态良好的3T3细胞(来自清华大学童佩老师)，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OD 492nm的吸光度。OD 492nm的吸光度可以反应胶原蛋白或其片段的细胞粘附活性。蛋白的细胞粘附活性越高，越能在短时间给细胞提供优质的外环境，帮助细胞贴壁。
结果参见图8至图10，图8至图10基于三次平行实验的OD 492nm平均值和标准误作图。
图8至图10的结果表明，三种人重组胶原蛋白(即C17A3、C17B3、C17C1)与商品化的人胶原蛋白相比，皆具有很好的细胞粘附活性。
序列
SEQ ID NO.1(C17A)
Figure PCTCN2020123600-appb-000012
SEQ ID NO.2(C17B)
Figure PCTCN2020123600-appb-000013
SEQ ID NO.3(C17C1)
Figure PCTCN2020123600-appb-000014
SEQ ID NO.4(C17A3)
Figure PCTCN2020123600-appb-000015
SEQ ID NO.5(C17A3-DNA)
Figure PCTCN2020123600-appb-000016
SEQ ID NO.6(C17B3)
Figure PCTCN2020123600-appb-000017
SEQ ID NO.7(C17B3-DNA)
Figure PCTCN2020123600-appb-000018
SEQ ID NO.8(C17C1-DNA)
Figure PCTCN2020123600-appb-000019
Figure PCTCN2020123600-appb-000020
SEQ ID NO.9(COL17A1)
Figure PCTCN2020123600-appb-000021
Figure PCTCN2020123600-appb-000022

Claims (10)

  1. 多肽，所述多肽包含SEQ ID No.9中的63至1496个连续的氨基酸残基，其中所述多肽具有细胞粘附活性。
  2. 多肽，其中所述多肽包含(A) m所示的序列或者由(A) m所示的序列组成，其中每个A为选自SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个所示的氨基酸序列或SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个取代、添加、或缺失了1或多个，例如2、3、4或5个氨基酸残基的氨基酸序列或者与SEQ ID No.1、SEQ ID No.2和SEQ ID No.3任一个所示的氨基酸序列具有83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％或97％序列同一性的序列；m为1-10之间的整数；其中每个A相同或不同并且相邻两个A之间直接通过肽键连接或者通过1个以上的氨基酸残基连接；其中所述多肽具有细胞粘附活性。
  3. 权利要求1或2所述的多肽，其中所述多肽包含SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.6所示的氨基酸序列或由其组成。
  4. 多核苷酸，其编码根据权利要求1-3中任一项所述的多肽，优选地，所述多核苷酸包含SEQ ID No.5、SEQ ID No.7、或SEQ ID No.8所示的核苷酸序列或由SEQ ID No.5、SEQ ID No.7、或SEQ ID No.8所示的核苷酸序列组成。
  5. 表达载体，其包含根据权利要求4所述的多核苷酸。
  6. 宿主细胞，其包含根据权利要求5所述的表达载体或者表达权利要求1-3中任一项所述的多肽，其中所述宿主细胞优选是大肠杆菌细胞。
  7. 制备根据权利要求1-3中任一项所述的多肽的方法，其包括：
    (1)在生产培养基中培养根据权利要求6所述的宿主细胞；
    (2)从宿主细胞分离出权利要求1-3中任一项所述的多肽。
  8. 组合物，其包含根据权利要求1-3中任一项所述的多肽或权利要求7的方法制备的多肽。
  9. 制品，其包含根据权利要求1-3中任一项所述的多肽或权利要求7的方法制备的多肽或者权利要求8所述的组合物，其中所述制成品为药物组合物、医疗器材、组织工程产品、化妆品或保健品，优选地所述药物组 合物是外用制剂，优选外用涂抹制剂，例如外用凝胶剂或外用浸润制剂；其中优选地所述外用凝胶剂还包含药学上可接受的载体，所述外用浸润制剂还包含无菌医用棉球。
  10. 根据权利要求1-3中任一项所述的多肽或权利要求7的方法制备的多肽、权利要求4的多核苷酸、权利要求5的表达载体、权利要求6的宿主细胞、或权利要求8的组合物在制备制成品，优选医疗器材、组织工程产品、化妆品、保健品中的用途。
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CN116640231A (zh) * 2023-06-09 2023-08-25 广州暨南大学医药生物技术研究开发中心有限公司 一种重组人源化17型胶原多肽及其制备方法
CN116640231B (zh) * 2023-06-09 2024-03-26 广州暨南大学医药生物技术研究开发中心有限公司 一种重组人源化17型胶原多肽及其制备方法

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