WO2023231489A1 - 一种生物合成人体结构性材料的制备方法 - Google Patents

一种生物合成人体结构性材料的制备方法 Download PDF

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WO2023231489A1
WO2023231489A1 PCT/CN2023/079951 CN2023079951W WO2023231489A1 WO 2023231489 A1 WO2023231489 A1 WO 2023231489A1 CN 2023079951 W CN2023079951 W CN 2023079951W WO 2023231489 A1 WO2023231489 A1 WO 2023231489A1
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amino acid
collagen
acid sequence
protein
recombinant
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PCT/CN2023/079951
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English (en)
French (fr)
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金雪坤
兰小宾
王玲玲
张永建
杨霞
何振瑞
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山西锦波生物医药股份有限公司
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Publication of WO2023231489A1 publication Critical patent/WO2023231489A1/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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A61L15/325—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • 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 field of biotechnology, and specifically relates to a method for preparing biosynthetic human body structural materials.
  • Structural materials of the human body are mainly structural proteins including collagen. This type of protein has adhesion and support functions to cells and tissues and is the main component of the extracellular matrix.
  • Collagen is a type of protein widely distributed in human connective tissue. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Currently, it is found that there are at least 28 collagen subtypes in the human body, which are located in different tissues. organ. Among them, type IV collagen mainly exists in the hepatic portal vein vascular area, around the central vein, and is distributed along the sinusoidal space. It is an important component of the basement membrane skeleton. It has a non-fibrous mesh collagen triple helix structure and plays an important role in the extracellular matrix. It has the ability to support cells. Collagen is a natural biological resource with biological tissue compatibility unmatched by other polymer materials. Therefore, it can be used as an important structural material for the human body and is widely used in the high-end pharmaceutical industry.
  • recombinant humanized collagen refers to the full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombinant technology, or It is a combination containing functional fragments of human collagen.
  • type IV collagen is a non-fibrillar triple helix structure with six genetically different ⁇ chains: ⁇ 1(IV)- ⁇ 6(IV), which can form three different molecular forms in tissues. , namely ⁇ 1 ⁇ 1 ⁇ 2(IV), ⁇ 3 ⁇ 4 ⁇ 5(IV) and ⁇ 5 ⁇ 5 ⁇ 6(IV), which can be selectively expressed in different membranes in specific tissues at different stages. Structurally speaking, the structure of the human body's natural type IV collagen is very complex, which makes humanized collagen extremely difficult to express and prepare in large quantities through conventional means.
  • the synthesis and modification of collagen starts from procollagen and undergoes many chemical changes such as hydroxylation, glycosylation, and cross-linking, and is complexly regulated by a variety of biological enzymes.
  • tropocollagen also contains globular heads and tails. Without these heads and tails, the collagen chains do not fold into the correct triple helices, thus lacking the biological activity of the collagen. Therefore, collagen prepared according to the original gene sequence cannot spontaneously organize to form the correct spatial structure in vitro. Such difficulties seriously hinder the development and production of humanized collagen.
  • the traditional method of producing collagen is to use acid, alkali, and enzymatic hydrolysis to process animal-derived tissues to extract collagen derivatives.
  • the collagen extracted by these methods has lost its original biological activity and cannot be used in the biomedical field to perform real functions.
  • people continue to try to use transgenic technology to prepare recombinant human collagen in animal, plant and microbial expression systems, which solves many shortcomings of traditional extraction processes.
  • receptors that can be expressed in human B cells are not expressed in mice, indicating that humanized models can provide expression that cannot be obtained using conventional mice. Therefore, there is an urgent need for a biosynthetic method of humanized type IV collagen that can overcome this defect so that it can be widely used as a structural material for the human body.
  • the present invention intends to provide a recombinant type IV humanized collagen, its biosynthetic preparation method, and its use in the medical field to solve the problem that natural human type IV collagen cannot be correctly expressed in vitro and prepared in large quantities. , for the production of humanized collagen Applications provide new possibilities.
  • a first aspect of the present invention provides a recombinant type IV humanized collagen.
  • the present invention screens the sequence of the functional region of human natural type IV collagen and uses it as a repeating sequence, so that the amino acid sequence of the recombinant type IV humanized collagen contains n repeating sequences, that is, human natural IV Functional region sequence of type collagen, n is an integer greater than or equal to 1.
  • Recombinant humanized type IV collagen consists of a sequence of the functional domain of natural human type IV collagen.
  • n can be 1, 2, 3, 4, 5, 6, 7 or 8, wherein when n is an integer greater than or equal to 2, the repeating sequences are directly connected; thus, the amino acid sequence can be obtained Recombinant type IV humanized collagen with the sequence shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.
  • amino acid sequence of the recombinant type IV humanized collagen provided by the present invention can also include amino acids that can be cleaved by TEV protease.
  • amino acid sequence that can be cleaved by TEV protease is shown in SEQ ID NO.7; optionally, the amino acid sequence that can be cleaved by TEV protease is directly connected to the repeat sequence; preferably, the amino acid sequence that can be cleaved by TEV protease is directly connected.
  • the amino acid sequence cleaved by TEV protease is located at the N-terminus of the recombinant type IV humanized collagen.
  • the present invention also provides recombinant type IV humanized collagen with the above amino acid sequence as the core region.
  • a second aspect of the invention provides a polynucleotide encoding the recombinant type IV humanized collagen described in the first aspect of the invention.
  • sequence of the polynucleotide may include the sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or its derivatives based on artificial design and codon optimization.
  • the third aspect of the invention provides a series of recombinant expression vectors comprising the polynucleotides described in the second aspect of the invention.
  • the expression vector selected by the present invention can exist stably in various hosts of prokaryotic or eukaryotic cells and can replicate autonomously, such as conventional plasmids (pET series) in the field, shuttle vector PNV 18.1, phage or viral vectors, etc.
  • the nucleotide sequence described in the second aspect of the present invention is cloned into the vector through molecular biology operations such as enzyme digestion and ligation to construct a recombinant expression vector.
  • the fourth aspect of the present invention provides a series of recombinant host cells containing the recombinant expression vector described in the third aspect of the present invention.
  • the host cells are selected from prokaryotic cells, yeast or eukaryotic cells, and may further be selected from Escherichia coli and Rhodococcus erythrococcus. , Bacillus subtilis, yeast.
  • the recombinant expression vector described in the third aspect of the present invention is transformed into host cells to obtain corresponding genetically engineered bacteria.
  • the fifth aspect of the present invention provides a method for preparing recombinant type IV humanized collagen described in the first aspect of the present invention, which may include the following steps:
  • S1 Fermentation and culture of the recombinant host cells described in the fourth aspect of the present invention to induce protein expression; in this step, the fermentation culture can be carried out in a shaker with a rotation speed of 200-240 rpm and a temperature of 35-38°C.
  • the preferred rotation speed is 220 rpm, and the preferred temperature is 37°C;
  • the specific procedure for inducing protein expression can be: cooling to 16-30°C and adding IPTG.
  • the optional working concentration of IPTG is 0.3-0.7mM, preferably 0.5mM. ;
  • the specific procedure for collecting the protein can be: centrifuge the mixture obtained in step S1 at 5000-7000rpm for 10-15min at 4°C, and use the mixture containing 20-30mM Resuspend the pellet in an aqueous solution of Tris, 150-250mM sodium chloride and 15-25mM imidazole, homogenize with high pressure or disrupt the cells by ultrasonic, then centrifuge at 15000-18000rpm for 20-40min at 4°C; preferably, the protein collected is
  • the specific procedures available are: centrifuge the mixture obtained in step S1 at 6000 rpm for 12 minutes at 4°C, resuspend the pellet in an aqueous solution containing 25mM Tris, 200mM sodium chloride and 20mM imidazole, cool to ⁇ 15°C, and homogenize under high pressure or After ultrasonic disruption of the cells, centrifuge at 17,000 rpm for 30 minutes at 4°C;
  • S3 Purify the protein; preferably, this step includes using Ni affinity chromatography column and/or ion exchange chromatography column to purify the protein, and optionally enzymatically digesting the protein; preferably using TEV protease enzyme To cleave the protein, more preferably, the mass ratio of the protein to the TEV protease is (15-25):1.
  • the preparation method may also include the steps of screening the functional regions of human natural type IV collagen, the steps of constructing recombinant expression vectors, and the steps of constructing recombinant host cells; specifically, the The specific procedure of screening the functional region of human natural type IV collagen can be as follows: excluding uncharged amino acid motifs in the helical region of human natural type IV collagen, and then screening out interchain hydrogens through computer-assisted protein structure prediction. The potential helical functional region with the most bond structures and the best ability to stabilize the trimer aggregation form was then selected based on the prediction method of protein expression properties to select the human natural type IV protein with the highest expression amount, easy purification, and good stability.
  • Collagen functional region by repeatedly connecting the amino acid sequences of the functional regions of human natural type IV collagen obtained through screening and optionally adding amino acid sequences that can be cleaved by TEV protease, the method described in the first aspect of the present invention is obtained.
  • the amino acid sequence of the recombinant type IV humanized collagen can then be artificially designed and optimized according to the codon preference of the target organism.
  • the polynucleotide sequence of the gene encoding the above recombinant type IV humanized collagen can be obtained as described in the first step of the present invention.
  • the nucleotide sequence described in the second aspect is cloned into a vector to construct a recombinant expression vector as described in the third aspect of the present invention.
  • the above vector is transformed into a host cell to obtain a recombinant expression vector as described in the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides the use of the recombinant type IV humanized collagen described in the first aspect of the present invention in the preparation of products, including medical devices, cosmetics, health products or drugs.
  • the present invention is as follows:
  • a recombinant type IV humanized collagen wherein the amino acid sequence of the recombinant type IV humanized collagen contains n repeating sequences, n is an integer greater than or equal to 1; wherein, the repeating sequences It is the functional region sequence of human natural type IV collagen;
  • n 1, 2, 3, 4, 5, 6, 7 or 8, wherein when n is an integer greater than or equal to 2, the repeating sequences are directly connected;
  • amino acid sequence of the recombinant type IV humanized collagen includes any one of the following (i)-(iii):
  • amino acid sequence encoded by a nucleotide sequence that is consistent with a polynucleotide sequence encoding a sequence shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3 under stringent conditions hybridizes under low conditions, and the amino acid sequence retains the cell adhesion effect of human native type IV collagen, and the stringent conditions are moderately stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.
  • the amino acid sequence cleaved by TEV protease is shown in SEQ ID NO.7;
  • the amino acid sequence capable of being cleaved by TEV protease is directly connected to the repeat sequence;
  • the amino acid sequence capable of being cleaved by TEV protease is located at the N-terminus of the recombinant type IV humanized collagen.
  • sequence of the polynucleotide includes the sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.
  • a recombinant expression vector wherein the recombinant expression vector contains the polynucleotide described in [3];
  • the recombinant expression vector includes pET series vectors, shuttle vectors, phage or viral vectors;
  • the recombinant expression vector is pET-28a-Trx-His.
  • a recombinant host cell wherein the recombinant host cell contains the recombinant expression vector described in [4];
  • the recombinant host cell is a prokaryotic cell, yeast or eukaryotic cell;
  • the host cell is E. coli BL21 (DE3).
  • the preparation method of recombinant type IV humanized collagen described in [6], [1] or [2] includes the following steps:
  • S3 Purify the protein; preferably, it includes using Ni affinity chromatography column and/or ion exchange chromatography column to purify the protein, and optionally enzymatically digesting the protein, preferably using TEV protease to digest the protein.
  • the protein more preferably, the mass ratio of the protein to the TEV protease is (15-25):1.
  • the specific procedure for collecting the protein is: Centrifuge the mixture obtained in step S1 at 5000-7000rpm for 10-15min at 4°C, resuspend the pellet in an aqueous solution containing 20-30mM Tris, 150-250mM sodium chloride and 15-25mM imidazole, and homogenize under high pressure or ultrasonically disrupt it. The cells were then centrifuged at 15000-18000 rpm for 20-40 minutes at 4°C.
  • the present invention provides the amino acid sequence of recombinant humanized type IV collagen that can be correctly expressed in vitro and successfully biosynthesized in vitro.
  • the amino acid sequence of this protein is derived from natural human type IV collagen.
  • the protein will not produce adverse immune reactions when used in the human body, and compared with commercial humanized collagen, the recombinant type IV humanized collagen prepared by the present invention has a higher cell adhesion effect.
  • the preparation method provided by the invention is simple, and high-yield recombinant type IV humanized collagen can be obtained at low cost.
  • Figure 1 shows the electrophoresis detection results involved in the expression and purification process of recombinant type IV humanized collagen C007.
  • Figure 2 shows the electrophoresis detection results involved in the expression and purification process of recombinant type IV humanized collagen C4P7C6.
  • Figure 3 shows the electrophoresis detection results involved in the expression and purification process of recombinant type IV humanized collagen C4P7E5.
  • Figure 4 shows the comparison results of the expression, purification and stability of recombinant type IV humanized collagen C4P7C6 and recombinant type IV humanized collagen C007.
  • Figure 5 shows the comparison results of cell adhesion activities of different recombinant type IV humanized collagens.
  • Figure 6 is the vector map of the pET-28a-Trx-His expression vector.
  • the meaning expressed by "can” includes both the meaning of performing certain processing and not performing certain processing.
  • the terms “comprising”, “having”, “includes” or “containing” may mean inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Meanwhile, “comprises,” “having,” “includes” or “containing” can also mean closed terms, excluding additional, unrecited elements or method steps.
  • the numerical range expressed using “numeric value A to numerical value B", “above numerical value A” and “below numerical value A” means a range including the endpoint numerical values A and B.
  • any numerical value includes the standard deviation of the error of the device or method used to determine the value.
  • the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments are presented as accurately as possible. Any numerical value, however, inherently contains standard deviations resulting from any aforementioned testing equipment or methods.
  • polypeptide and “protein” interchangeably refer to a string of at least two amino acid residues connected to each other through covalent bonds (such as peptide bonds), which can be recombinant polypeptides, natural polypeptides or synthetic polypeptides.
  • a polypeptide can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
  • amino acid may include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers.
  • amino acid deletion may refer to deleting 1, 2 or 3 or more amino acids from an amino acid sequence, as long as the changed sequence completely or partially retains the activity of the original amino acid sequence.
  • amino acid addition may refer to adding 1, 2 or 3 or more amino acids at any position between the C-terminal, N-terminal or between the C-terminal and N-terminal of the amino acid sequence, as long as the changed sequence completely or partially retains the original activity of the amino acid sequence.
  • amino acid substitution may refer to the replacement of an amino acid at a certain position of an amino acid sequence by another amino acid, as long as the change The sequence completely or partially retains the activity of the original amino acid sequence.
  • Amino acid substitution can be conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or similar properties to form a peptide (conservative variant peptide).
  • these conservative variant peptides can be produced based on the following amino acid substitutions: substitution of Val, Leu or Ile for Ala, substitution of Lys, Gln, Asn or His for Arg, substitution of Gln, His, Lys or Arg for Asn , Substitution of Glu or Asn for Asp, Substitution of Ser or Ala for Cys, Substitution of Asn or Glu for Gln, Substitution of Asp or Gln for Glu, Substitution of Ala for Gly, Substitution of Asn, Lys, Gln or Arg for His.
  • substitution of Leu, Met, Ala, Val or Phe for Ile substitution of Ile, Met, Ala, Val or Phe for Leu, substitution of Asn, Gln or Arg for Lys, substitution of Ile, Leu or Phe for Met, Leu , Val, Ile, Ala or Tyr for Phe, Ala for Pro, Thr for Ser, Ser or Val for Thr, Phe or Tyr for Trp, Trp, Phe, Thr or Ser for Tyr Substitution of Val, and substitution of Val by Phe, Ala, Met, Ile or Leu.
  • Amino acid substitutions may also be non-conservative amino acid substitutions.
  • amino acid modifications may include modifications to natural sequences, such as modification of functional groups, intramolecular covalent bonding (for example, ring formation between side chains), methylation, acylation, ubiquitination, phosphorylation , aminohexanation, biotinylation, etc.
  • hybridize means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions without non-specific binding to non-complementary partners under these conditions.
  • sequences are preferably 90 to 100% complementary.
  • the properties of complementary sequences that can specifically bind to each other are used, for example, in Northern or Southern blotting techniques, or in primer binding for PCR or RT-PCR.
  • hybridization occurs under conditions of moderate stringency, medium-high stringency, high stringency or very high stringency. Such hybridization conditions are described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • specific hybridization conditions are as follows: (1) Low stringency hybridization conditions in 6 ⁇ sodium chloride/sodium citrate (SSC) at approximately 45°C, then at least 50°C in 0.2 ⁇ SSC, 0.1% SDS Medium wash 2 times (for low stringency conditions, the washing temperature can be increased to 55°C); (2) Medium stringency hybridization conditions are at 6 ⁇ SSC, at about 45°C, then at 60°C, at 0.2 ⁇ SSC, Wash 1 or more times in 0.1% SDS; (3) High stringency hybridization conditions in 6 ⁇ SSC, at about 45°C, then at 65°C, wash 1 or more times in 0.2 ⁇ SSC, 0.1% SDS and Preferred; (4) Very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 65°C in 0.2 ⁇ SSC, 1% SDS.
  • SSC sodium chloride/sodium citrate
  • suitable vectors are known in the art of vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vectors of the present invention include sequences suitable for introduction into cells.
  • the vector can be an expression vector, in which the coding sequence of the protein is controlled by its own cis-acting regulatory elements, and the vector is designed to facilitate gene integration or gene replacement in the host cell.
  • "vector” includes DNA molecules, such as plasmids, phages, viruses, or other vectors, which contain one or more heterologous or recombinant nucleotide sequences.
  • Suitable phages and viral vectors include, but are not limited to: lambda-phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine breast cancer virus, lentivirus, etc. .
  • the host cell may be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Enterobacteriaceae.
  • the "functional region of human natural type IV collagen” refers to a sequence fragment in human natural type IV collagen ⁇ 1 that has functions such as cell adhesion activity possessed by the collagen amino acid sequence.
  • the application fields of the recombinant type IV humanized collagen provided by the present invention include the preparation of high-end medical devices, such as biological dressings, human bionic materials, plastic surgery materials, organoid culture, cardiovascular stents, coatings, tissue injection filling, and ophthalmic materials. , obstetrics and gynecology biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, 3D printing artificial organ biomaterials, etc.; as well as high-end cosmetic raw materials, high-end health products and high-end pharmaceutical excipients, etc.
  • high-end medical devices such as biological dressings, human bionic materials, plastic surgery materials, organoid culture, cardiovascular stents, coatings, tissue injection filling, and ophthalmic materials. , obstetrics and gynecology biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, 3D printing artificial organ biomaterials, etc.; as well as high-end cosmetic raw materials, high-end health products and
  • Example 1 Construction and expression of recombinant type IV humanized collagen
  • the amino acid sequence ENLYFQ (SEQ ID NO. 7) that can be cleaved by TEV protease can be added to its N terminus.
  • the amino acid sequence that can be cleaved by TEV protease The sequence is directly linked to the functional region sequence of the human native type IV collagen.
  • e. Wash the column with 1M imidazole working solution at a flow rate of 10mL/min, and perform electrophoresis detection on the flow-through (sample name is recorded as "1M wash").
  • B solution contains 1M sodium chloride and 20mM Tris. The above percentage is the volume fraction. The peaks are collected and analyzed by electrophoresis (sample name is recorded as " B elution"). d. Clean the column material. e. Detect the target protein content and calculate the protein yield, and store the protein in a 4°C environment.
  • Test results The electrophoresis detection results involved in the preparation process of each recombinant protein are shown in Figures 1 to 3.
  • Figure 4 shows the comparison of the expression stability of recombinant type IV humanized collagen C4P7C6 and C007. in:
  • Figure 3 shows the electrophoresis test results of recombinant type IV humanized collagen C4P7E5. It can be seen that the purification and enzyme digestion effects are good.
  • the principle of this method is to measure the characteristic absorption of peptide bonds under far ultraviolet light, which is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for the detection of human collagen and its analogues that do not develop color in Coomassie brilliant blue. . (The reference is Walker JM. The Protein Protocols Handbook, second edition. HumanaPress.43-45.). After detecting the protein concentration, use PBS to adjust the concentration of all proteins to be tested to 0.5 mg/mL.
  • cell adhesion rate (test well-blank well)/(positive well-blank well) ⁇ 100%.
  • the cell adhesion rate can reflect the activity of collagen. The higher the activity of the protein, the better it can provide cells with a high-quality external environment in a short period of time and help cells adhere to the wall.
  • the recombinant type IV humanized collagen of the present invention has better bioadhesion activity, that is: recombinant type IV humanized collagen Protein C4P7C6>C007>C4P7E5>Human collagen.
  • Example 3 Mass spectrometry detection of recombinant type IV humanized collagen
  • the protein identification results are obtained based on the primary mass spectrometry of the peptide fragments produced after enzymatic digestion. Detection parameters: Trypsin enzymatic hydrolysis, with two missed cleavage sites. Set alkylation of cysteine as a fixed modification. Oxidation of methionine is a variable modification.
  • the database used for identification is NCBprot.
  • the coverage rate of detected polypeptide fragments was 75.08%, and the detection results were very credible.
  • the coverage rate of detected polypeptide fragments was 100%, and the detection results were very reliable.
  • the coverage rate of detected polypeptide fragments was 100%, and the detection results were very reliable.

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Abstract

本发明公开了一种生物合成人体结构性材料的制备方法。本发明提供了一种重组IV型人源化胶原蛋白，其氨基酸序列包含n个重复序列，n为大于等于1的整数；其中，所述重复序列为人天然IV型胶原蛋白的功能区序列。本发明提供了能够在体外正确表达的重组IV型人源化胶原蛋白的氨基酸序列，并且成功在体外进行了生物合成，该蛋白的氨基酸序列源于天然人IV型胶原蛋白，用于人体不会产生不良免疫反应，并且相比商品化的人源胶原蛋白，本发明制备的重组IV型人源化胶原蛋白具有更高的细胞黏附效果。同时，本发明提供的制备方法简单，在低成本下即可获得较高产量的重组IV型人源化胶原蛋白。

Description

一种生物合成人体结构性材料的制备方法
优先权和相关申请
本申请要求于2022年6月1日提交中国专利局、申请号为202210619454.1、发明名称为“一种生物合成人体结构性材料的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及一种生物合成人体结构性材料的制备方法。
背景技术
人体结构性材料主要是包含胶原蛋白的结构性蛋白，这类蛋白对细胞、组织具有黏附、支撑功能，是主要的细胞外基质组成成分。
胶原蛋白是广泛分布于人体结缔组织的一类蛋白质，也是人体含量最多的蛋白质，可以占到蛋白质总量的25％～35％，目前发现人体至少有28种胶原蛋白亚型，分别位于不同组织器官。其中，Ⅳ型胶原蛋白主要存在于肝门静脉血管区，中央静脉周围，沿着窦状隙分布，是构成基底膜骨架的重要成分，其呈非纤维网状胶原蛋白三螺旋结构，发挥细胞外基质作用，对细胞有支撑能力。胶原蛋白是天然的生物资源，具有其它高分子材料望尘莫及的生物组织相容性，因此可以作为重要的人体结构性材料，广泛的应用在高端医药行业。
然而，目前胶原蛋白原材料主要源自于动物组织提取，遗憾的是，Ⅳ型胶原蛋白在动物体内含量低，致使无法提取到Ⅳ型胶原蛋白单一成分。同时，动物源性免疫反应也是导致胶原蛋白在应用中受限的重要原因。随着我国胶原蛋白产业的日益壮大，利用生物合成路径获得胶原蛋白日趋成熟，尤其是人源化胶原蛋白已经走在世界前列。2021年国家药监局对生物合成胶原蛋白做了命名分类，其中，重组人源化胶原蛋白是指由DNA重组技术制备的人胶原蛋白特定型别基因编码的全长或部分氨基酸序列片段，或是含人胶原蛋白功能片段的组合。
在生物体中，Ⅳ型胶原蛋白是一种非纤维状三螺旋结构，有六种遗传上不同的α链：α1(Ⅳ)-α6(Ⅳ)，会在组织中形成三种不同的分子形式，即α1α1α2(Ⅳ)、α3α4α5(Ⅳ)和α5α5α6(Ⅳ)，它们能够在不同阶段的特定组织中选择性的在不同的膜中表达。从结构上来说，人体天然的Ⅳ型胶原蛋白的结构非常的复杂，所以才导致人源化胶原蛋白极难通过常规手段表达和大量制备。胶原蛋白的合成和修饰从原胶原开始，经历了羟基化、糖基化、相互交联等诸多化学变化，受到了多种生物酶的复杂调控。原胶原除了含有胶原链之外，还含有球状的头部和尾部。没有这些头部和尾部，胶原链就不会折叠成为正确的三螺旋，从而缺乏胶原蛋白的生物学活性。因此，按照原始基因序列制备的胶原蛋白无法在体外自发的组织形成正确的空间结构。这样的困难严重阻碍了人源化胶原蛋白的研发和生产。
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。随着现代生物技术的发展，人们不断尝试利用转基因技术，在动物、植物和微生物表达体系中制备重组人胶原蛋白，解决了传统提取工艺的诸多缺点。但是也有研究机构指出，人体B细胞中可表现出来的受体在小鼠中未表达出来，所以表明人源化的模型可以提供使用常规小鼠无法获得的表达。所以亟需一种可以克服该缺陷的人源化Ⅳ型胶原蛋白生物合成方法，使其可作为人体结构性材料得到广泛应用。
发明内容
发明要解决的问题
本发明拟提供一种重组IV型人源化胶原蛋白，并提供其生物合成制备方法，及其在医药领域的用途，用以解决天然人Ⅳ型胶原蛋白在体外无法正确表达并大量制备的问题，为人源化胶原蛋白的生产 应用提供新的可能。
用于解决问题的方案
本发明的第一方面提供一种重组IV型人源化胶原蛋白。具体来说，本发明筛选了人天然IV型胶原蛋白的功能区的序列，并将其作为重复序列，使所述重组IV型人源化胶原蛋白的氨基酸序列包含n个重复序列即人天然IV型胶原蛋白的功能区序列，n为大于等于1的整数。重组IV型人源化胶原蛋白由一种人天然IV型胶原蛋白的功能区的序列组成。
进一步的，所述n可以为1、2、3、4、5、6、7或8，其中，当n为大于等于2的整数时，各重复序列之间直接连接；从而可以获得氨基酸序列包含如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示序列的重组IV型人源化胶原蛋白。
更进一步的，为了方便所述重组IV型人源化胶原蛋白在实际生产过程中的表达和纯化，本发明提供的重组IV型人源化胶原蛋白的氨基酸序列还可以包含能够通过TEV蛋白酶切除的氨基酸序列，所述能够通过TEV蛋白酶切除的氨基酸序列如SEQ ID NO.7所示；可选的，所述能够通过TEV蛋白酶切除的氨基酸序列与所述重复序列直接连接；优选的，所述能够通过TEV蛋白酶切除的氨基酸序列位于所述重组IV型人源化胶原蛋白的N末端。
本发明还提供了以上述氨基酸序列为核心区域的重组IV型人源化胶原蛋白。
本发明的第二方面提供编码本发明第一方面中所述重组IV型人源化胶原蛋白的多核苷酸。
进一步的，所述多核苷酸的序列可以包含如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示的序列，或其根据人工设计和密码子优化后的衍生物。
本发明的第三方面提供一系列包含本发明第二方面中所述多核苷酸的重组表达载体。本发明选择的表达载体可以在原核或真核细胞的各种宿主中稳定存在并可自主复制，如本领域中的常规质粒(pET系列)、穿梭载体PNV 18.1、噬菌体或病毒载体等。在载体上通过酶切、连接等分子生物学操作，将本发明第二方面中所述的核苷酸序列克隆到载体中，构建得到重组表达载体。
本发明的第四方面提供一系列包含本发明第三方面中所述重组表达载体的重组宿主细胞，所述宿主细胞选自原核细胞、酵母或真核细胞，进一步可以选自大肠杆菌、赤红球菌、枯草芽孢杆菌、酵母菌。将本发明第三方面中所述的重组表达载体转化至宿主细胞中，获得相应的基因工程菌。
本发明的第五方面提供本发明第一方面中所述重组IV型人源化胶原蛋白的制备方法，其可以包括如下步骤：
S1：发酵培养本发明第四方面所述的重组宿主细胞，诱导蛋白表达；在该步骤中，所述发酵培养可选在转速为200-240rpm、温度为35-38℃的摇床中进行，优选转速为220rpm，优选温度为37℃；所述诱导蛋白表达的具体程序可选为：降温至16-30℃并添加IPTG，可选的IPTG的工作浓度为0.3-0.7mM，优选为0.5mM；
S2：收集所述蛋白；在该步骤中，所述收集所述蛋白的具体程序可选为：将S1步骤所得到的混合物于4℃环境下5000-7000rpm离心10-15min，利用含有20-30mM Tris、150-250mM氯化钠和15-25mM咪唑的水溶液重悬沉淀，高压均质或超声破碎细胞后于4℃环境下15000-18000rpm离心20-40min；优选的，所述收集所述蛋白的具体程序可选为：将S1步骤所得到的混合物于4℃环境下6000rpm离心12min，利用含有25mM Tris、200mM氯化钠和20mM咪唑的水溶液重悬沉淀，降温至≤15℃，高压均质或超声破碎细胞后于4℃环境下17000rpm离心30min；
S3：纯化所述蛋白；优选的，该步骤包括使用Ni亲和层析柱和/或离子交换层析柱纯化所述蛋白，以及任选的对所述蛋白进行酶切；优选使用TEV蛋白酶酶切所述蛋白，更优选的，所述蛋白与所述TEV蛋白酶的质量比为(15-25):1。
进一步的，在所述S1步骤前，所述制备方法还可以包括筛选人天然IV型胶原蛋白的功能区的步骤、重组表达载体的构建步骤、重组宿主细胞的构建步骤；具体而言，所述筛选人天然IV型胶原蛋白的功能区的步骤的具体程序可以为：排除人天然IV型胶原蛋白螺旋区中不含电荷的氨基酸基序，随后通过计算机辅助蛋白结构预测的方法筛选出链间氢键结构最多、最能稳定三聚体聚集形式的潜在螺旋功能区，随后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，稳定性好的人天然IV型 胶原蛋白功能区域；通过对筛选得到的人天然IV型胶原蛋白的功能区的氨基酸序列进行重复连接并任选的添加能够通过TEV蛋白酶切除的氨基酸序列，获得如本发明第一方面中所述的重组IV型人源化胶原蛋白的氨基酸序列，随后可以根据目标生物的密码子偏好性人工设计和优化上述重组IV型人源化胶原蛋白的编码基因的多核苷酸序列，从而获得如本发明第二方面中所述的核苷酸序列，将其克隆到载体中，构建得到如本发明第三方面所述的重组表达载体，将上述载体转化至宿主细胞中，获得如本发明第四方面所述的重组宿主细胞。
本发明的第六方面提供本发明第一方面中所述重组IV型人源化胶原蛋白在制备产品中的用途，所述产品包括医疗器械、化妆品、保健品或药物。
具体而言，本发明如下所述：
[1]、一种重组IV型人源化胶原蛋白，其中，所述重组IV型人源化胶原蛋白的氨基酸序列包含n个重复序列，n为大于等于1的整数；其中，所述重复序列为人天然IV型胶原蛋白的功能区序列；
由选的，所述n为1、2、3、4、5、6、7或8，其中，当n为大于等于2的整数时，各重复序列之间是直接连接的；
更优选的，所述重组IV型人源化胶原蛋白的氨基酸序列包含如下(i)-(iii)中的任一项：
(i)如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的氨基酸序列；
(ii)在如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的氨基酸序列中添加、取代、缺失或修饰1个或多个氨基酸残基并且保留人天然IV型胶原蛋白的细胞黏附效果的氨基酸序列；
(iii)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留人天然IV型胶原蛋白的细胞黏附效果，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。
[2]、根据[1]所述的重组IV型人源化胶原蛋白，其中，所述重组IV型人源化胶原蛋白的氨基酸序列还包含能够通过TEV蛋白酶切除的氨基酸序列，所述能够通过TEV蛋白酶切除的氨基酸序列如SEQ ID NO.7所示；
优选的，所述能够通过TEV蛋白酶切除的氨基酸序列与所述重复序列直接连接；
优选的，所述能够通过TEV蛋白酶切除的氨基酸序列位于所述重组IV型人源化胶原蛋白的N末端。
[3]、一种编码[2]所述的重组IV型人源化胶原蛋白的多核苷酸；
优选的，所述多核苷酸的序列包含如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的序列。
[4]、一种重组表达载体，其中，所述重组表达载体包含[3]所述的多核苷酸；
优选的，所述重组表达载体包括pET系列载体、穿梭载体、噬菌体或病毒载体；
更优选的，所述重组表达载体为pET-28a-Trx-His。
[5]、一种重组宿主细胞，其中，所述重组宿主细胞包含[4]所述的重组表达载体；
优选的，所述重组宿主细胞为原核细胞、酵母或真核细胞；
更优选的，所述宿主细胞为大肠杆菌BL21(DE3)。
[6]、[1]或[2]所述的重组IV型人源化胶原蛋白的制备方法，其包括以下步骤：
S1：发酵培养根据权利要求5所述的重组宿主细胞，诱导蛋白表达；
S2：收集所述蛋白；
S3：纯化所述蛋白；优选的，包括使用Ni亲和层析柱和/或离子交换层析柱纯化所述蛋白，以及任选的对所述蛋白进行酶切，优选用TEV蛋白酶酶切所述蛋白，更优选的，所述蛋白与所述TEV蛋白酶的质量比为(15-25):1。
[7]、根据[6]所述的制备方法，其中，在所述S1步骤中，所述发酵培养在转速为200-240rpm、温度为35-38℃的摇床中进行。
[8]、根据[6]或[7]所述的制备方法，其中，在所述S1步骤中，所述诱导蛋白表达的具体程序为：降温至16-30℃并添加IPTG；优选的，所述IPTG的工作浓度为0.3-0.7mM。
[9]、根据[6]-[8]中任一项所述的制备方法，在所述S2步骤中，所述收集所述蛋白的具体程序为： 将S1步骤所得到的混合物于4℃环境下5000-7000rpm离心10-15min，利用含有20-30mM Tris、150-250mM氯化钠和15-25mM咪唑的水溶液重悬沉淀，高压均质或超声破碎细胞后于4℃环境下15000-18000rpm离心20-40min。
[10]、[1]或[2]所述的重组IV型人源化胶原蛋白在制备产品中的用途，所述产品包括医疗器械、化妆品、保健品或药物。
发明的效果
通过以上技术方案的实施，本发明提供了能够在体外正确表达的重组IV型人源化胶原蛋白的氨基酸序列，并且成功在体外进行了生物合成，该蛋白的氨基酸序列源于天然人IV型胶原蛋白，用于人体不会产生不良免疫反应，并且相比商品化的人源胶原蛋白，本发明制备的重组IV型人源化胶原蛋白具有更高的细胞黏附效果。同时，本发明提供的制备方法简单，在低成本下即可获得较高产量的重组IV型人源化胶原蛋白。
附图说明
图1为重组Ⅳ型人源化胶原蛋白C007在表达及纯化过程中所涉及到的电泳检测结果。
图2为重组Ⅳ型人源化胶原蛋白C4P7C6在表达及纯化过程中所涉及到的电泳检测结果。
图3为重组Ⅳ型人源化胶原蛋白C4P7E5在表达及纯化过程中所涉及到的电泳检测结果。
图4为重组Ⅳ型人源化胶原蛋白C4P7C6和重组Ⅳ型人源化胶原蛋白C007的表达纯化稳定情况对比结果。
图5为不同重组Ⅳ型人源化胶原蛋白的细胞黏附活性比较结果。
图6为pET-28a-Trx-His表达载体的载体图谱。
具体实施方式
以下对本发明的实施方式进行说明，但本发明不限定于此。
在本发明中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
在本发明中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。
在本发明中，术语“包含”、“具有”、“包括”或“含有”可以指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。与此同时，“包含”、“具有”、“包括”或“含有”也可以表示封闭式的，排除额外的、未引述的元件或方法步骤。
在本发明中，使用“数值A～数值B”、“在数值A以上”和“在数值A以下”表示的数值范围是指包含端点数值A、B的范围。
在本发明中，任意数值包括测定该值所使用的装置或方法的误差的标准偏差。用以界定本发明的数值范围与参数皆是约略的数值，此处已尽可能精确地呈现具体实施例中的相关数值。然而，任何数值本质上不可避免地含有因前述测试装置或方法所致的标准偏差。
在本发明中，术语“多肽”、“蛋白”可互换地指通过共价键(例如肽键)相互连接的一串至少两个氨基酸残基，可以是重组多肽、天然多肽或合成多肽。多肽可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。
在本发明中，术语“氨基酸”可以包括天然氨基酸、非天然氨基酸、氨基酸类似物以及所有它们的D和L立体异构体。
在本发明中，氨基酸缺失可指从氨基酸序列中删除1、2或3个以上氨基酸，只要改变后的序列完全或部分保留原有氨基酸序列的活性。
在本发明中，氨基酸添加可指在氨基酸序列的C端、N端或C端与N端中间的任意位置处添加1、2或3个以上氨基酸，只要改变后的序列完全或部分保留原有氨基酸序列的活性。
在本发明中，氨基酸取代可指在氨基酸序列的某个位置的氨基酸被其他氨基酸替代，只要改变后 的序列完全或部分保留原有氨基酸序列的活性。氨基酸取代可以是保守氨基酸取代，指与原有氨基酸序列相比，若干个氨基酸被性质相似或相近的氨基酸所取代而形成肽(保守性变异肽)。示例性的，这些保守性变异肽可以根据下列氨基酸取代而产生：Val、Leu或Ile对Ala的取代，Lys、Gln、Asn或His对Arg的取代，Gln、His、Lys或Arg对Asn的取代，Glu或Asn对Asp的取代，Ser或Ala对Cys的取代，Asn或Glu对Gln的取代，Asp或Gln对Glu的取代，Ala对Gly的取代，Asn、Lys、Gln或Arg对His的取代，Leu、Met、Ala、Val或Phe对Ile的取代，Ile、Met、Ala、Val或Phe对Leu的取代，Asn、Gln或Arg对Lys的取代，Ile、Leu或Phe对Met的取代，Leu、Val、Ile、Ala或Tyr对Phe的取代，Ala对Pro的取代，Thr对Ser的取代，Ser或Val对Thr的取代，Phe或Tyr对Trp的取代，Trp、Phe、Thr或Ser对Tyr的取代，及Phe、Ala、Met、Ile或Leu对Val的取代。氨基酸取代也可以是非保守氨基酸取代。
在本发明中，氨基酸修饰可以包括对天然序列的修饰，例如官能团的修饰、分子内共价键合(例如，侧链之间成环)、甲基化、酰基化、泛素化、磷酸化、氨基己烷化、生物素化等。
在本发明中，“杂交”意指多核苷酸或寡核苷酸在严格条件下与大体互补的序列结合的能力，而在这些条件下不发生与非互补对象之间的非特异性结合。对此，所述序列优选为90～100％互补的。将能够互相特异性结合的互补序列的特性应用于例如Northern或Southern印迹技术中，或是PCR或RT-PCR的引物结合中。根据本发明，杂交在中等严格条件、中-高严格条件、高严格条件或非常高严格条件条件下发生。此类杂交条件描述于描述于Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.(1989),6.3.1-6.3.6中。例如，具体的杂交条件如下：(1)低严格性杂交条件在6×氯化钠/柠檬酸钠(SSC)中，在约45℃，然后在至少50℃，在0.2×SSC，0.1％SDS中洗涤2次(对于低严格性条件，可以将洗涤温度升高到55℃)；(2)中等严格性杂交条件在6×SSC，在约45℃，然后在60℃，在0.2×SSC，0.1％SDS中洗涤1次或多次；(3)高严格性杂交条件在6×SSC，在约45℃，然后在65℃，在0.2×SSC，0.1％SDS中洗涤1次或多次且优选；(4)非常高的严格性杂交条件是0.5M磷酸钠，7％SDS，在65℃，然后在65℃，在0.2×SSC，1％SDS中洗涤1次或多次。
在本发明中，合适的载体是载体构建领域已知的，包括启动子的选择和其他调控元件，例如增强子元件。本发明所述的载体包括适合引入细胞的序列。例如，载体可以是表达载体，在该载体中，所述蛋白的编码序列受到它自身顺式作用调控元件的控制，载体的设计便于宿主细胞的基因整合或基因替换等。本领域普通技术人员应理解，在本发明中，“载体”包括DNA分子，例如，质粒、噬菌体、病毒或其他载体，它含有一个或多个异源的或重组的核苷酸序列。合适的噬菌体和病毒载体包括，但不限于：λ-噬菌体、EMBL噬菌体、猿猴病毒、牛疣病毒、Epstein-Barr病毒、腺病毒、疱疹病毒、小鼠肉瘤病毒、鼠类乳癌病毒、慢病毒等。
在本发明中，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。
在本发明中，“人天然IV型胶原蛋白的功能区”是指在人天然IV型胶原蛋白α1中有胶原蛋白氨基酸序列所具有的细胞粘附活性等功能的序列片段。
本发明提供的重组Ⅳ型人源化胶原蛋白的应用领域包含高端医疗器械制备，如生物敷料、人体仿生材料、整形美容材料、类器官培养、心血管支架、涂层、组织注射填充、眼科材料、妇产科生物材料、神经修复再生、肝脏组织及血管修复再生、3D打印人造器官生物材料等；以及高端化妆品原料、高端保健品和高端药用辅料等。
实施例
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。本发明的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所有试剂或仪器未注明生产厂商者，均为可以通过市购的常规产品。为了更好地说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实施例中，对于本领域技术人员熟知的方法、手段、器材和步骤未作详细描述，以便凸显本发明的主 旨。
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。如无特殊说明，本说明书中所使用的单位均为国际标准单位，并且本发明中出现的数值和数值范围，均应当理解为包含了不可避免的系统性误差。
实施例1：重组Ⅳ型人源化胶原蛋白构建及表达
1.进行大规模蛋白功能区筛选：1)序列筛选，在天然IV型胶原蛋白螺旋区的Gly-X-Y的重复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；2)借助计算机辅助蛋白结构预测的方法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；3)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IV型胶原蛋白功能区域；4)将筛选后得到的区域的氨基酸片段通过n次重复直接连接进行优化，以便保证重组IV型人源化胶原蛋白的分子量在一定范围，且易于纯化和稳定；最终得到以下不同的重组Ⅳ型人源化胶原蛋白的目的片段：
(1)重组Ⅳ型人源化胶原蛋白C007的氨基酸序列：GQKGDQGEKGQIGPIGEKGSRGDPGTPGVPGKDGQAGQPGQPGPKGDPGISGTPGAPGLPGPKGSVGGMGLPGTPGEKGVPGIPGPQGSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGEKGEKGSIGIPGMPGSPGLKGSPGSVGYPGSPGLPGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHATE(SEQ ID NO.1)；
(2)重组Ⅳ型人源化胶原蛋白C4P7C6的氨基酸序列：GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHA(SEQ ID NO.2)；
(3)重组Ⅳ型人源化胶原蛋白C4P7E5的氨基酸序列：GLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFM(SEQ ID NO.3)。
本发明的重组Ⅳ型人源化胶原蛋白在表达时，可以在其N末端添加能够被TEV蛋白酶切除的氨基酸序列ENLYFQ(SEQ ID NO.7)，优选的，所述能够通过TEV蛋白酶切除的氨基酸序列与所述人天然IV型胶原蛋白的功能区序列直接连接。
2.将合成的多核苷酸片段分别插入pET-28a-Trx-His表达载体(载体图谱如图6所示)中得到对应的重组表达质粒。其中，各重组Ⅳ型人源化胶原蛋白的多核苷酸片段如下所示：
(1)N末端添加能够被TEV蛋白酶切除的氨基酸序列的重组Ⅳ型人源化胶原蛋白C007的多核苷酸序列：GAAAACCTGTATTTCCAGGGTCAGAAAGGAGATCAAGGAGAAAAAGGACAGATAGGACCAATAGGAGAAAAAGGGAGCCGGGGAGATCCGGGAACACCGGGAGTGCCGGGAAAAGATGGACAGGCAGGCCAGCCGGGACAGCCGGGTCCTAAAGGAGATCCGGGTATAAGCGGAACACCGGGTGCACCGGGATTACCTGGTCCGAAAGGGAGCGTAGGGGGAATGGGACTGCCGGGAACACCTGGTGAAAAAGGCGTACCTGGTATTCCGGGTCCTCAGGGGTCCCCGGGACTGCCTGGTGATAAAGGTGCAAAAGGGGAAAAAGGGCAAGCAGGTCCGCCGGGTATTGGTATACCGGGTCTGCGTGGGGAAAAAGGTGATCAGGGTATTGCCGGTTTTCCGGGTAGCCCGGGTGAGAAAGGTGAAAAAGGAAGTATAGGAATACCGGGTATGCCTGGTAGTCCGGGTCTGAAAGGTAGCCCGGGAAGCGTGGGGTATCCGGGTAGCCCTGGTCTGCCTGGGGAAAAAGGCGATAAAGGTCTGCCGGGTCTGGACGGTATTCCTGGTGTTAAAGGGGAAGCAGGTCTGCCGGGGACCCCGGGTCCAACAGGTCCTGCAGGGCAGAAAG GTGAACCAGGTAGCGATGGTATTCCGGGGTCTGCGGGGGAAAAAGGAGAACCGGGTCTGCCGGGAAGAGGTTTTCCGGGCTTTCCGGGTGCAAAAGGAGATAAGGGTAGCAAAGGCGAAGTGGGTTTTCCGGGACTGGCAGGTAGTCCGGGAATCCCTGGTAGCAAAGGTGAACAGGGTTTTATGGGGCCGCCGGGTCCGCAGGGTCAGCCTGGTTTACCGGGTAGCCCAGGTCATGCGACAGAG(SEQ ID NO.4)；
(2)N末端添加能够被TEV蛋白酶切除的氨基酸序列的重组Ⅳ型人源化胶原蛋白C4P7C6的多核苷酸序列：GAAAATCTATATTTCCAAGGATTTCCCGGGTTCCCGGGTGCAAAAGGCGACAAGGGTTCGAAAGGCGAAGTTGGCTTCCCGGGATTAGCGGGTAGCCCAGGTATCCCGGGTTCTAAGGGTGAACAGGGTTTCATGGGTCCGCCCGGCCCACAGGGTCAACCGGGCCTGCCTGGTAGCCCGGGTCATGCGGGCTTTCCAGGTTTCCCGGGCGCGAAAGGCGACAAAGGTAGCAAGGGCGAAGTCGGTTTTCCGGGCCTTGCTGGCAGCCCGGGCATCCCGGGGTCCAAGGGCGAGCAAGGATTCATGGGTCCGCCTGGCCCACAAGGCCAACCGGGTTTGCCGGGCTCTCCGGGTCACGCCGGTTTTCCGGGATTCCCGGGAGCGAAAGGTGATAAGGGCTCCAAAGGTGAGGTGGGTTTTCCGGGCTTGGCTGGCAGCCCGGGTATTCCGGGTAGCAAGGGCGAGCAGGGTTTTATGGGCCCGCCTGGTCCGCAGGGCCAACCGGGGCTGCCGGGCTCCCCGGGCCACGCAGGCTTCCCAGGTTTTCCGGGTGCGAAAGGCGATAAAGGTAGTAAGGGTGAAGTGGGCTTCCCGGGTCTCGCGGGTAGCCCGGGTATTCCGGGCTCGAAGGGCGAACAGGGCTTTATGGGCCCACCGGGTCCGCAGGGTCAGCCGGGCCTGCCGGGTAGCCCGGGCCACGCGGGTTTCCCGGGCTTCCCGGGTGCCAAAGGCGATAAAGGTTCAAAGGGGGAGGTGGGTTTTCCGGGTCTGGCAGGCAGCCCGGGCATCCCGGGCAGCAAGGGCGAACAAGGTTTTATGGGTCCGCCTGGACCGCAAGGTCAGCCGGGCCTGCCGGGTAGTCCGGGCCATGCCGGCTTCCCGGGTTTTCCGGGTGCGAAAGGGGACAAAGGCAGCAAGGGAGAGGTTGGTTTCCCAGGCCTGGCTGGTAGCCCGGGTATTCCGGGCTCCAAGGGTGAGCAGGGTTTCATGGGTCCGCCTGGCCCACAGGGTCAACCTGGCCTGCCGGGTTCTCCGGGTCATGCG(SEQ ID NO.5)；
(3)N末端添加能够被TEV蛋白酶切除的氨基酸序列的重组Ⅳ型人源化胶原蛋白C4P7E5的多核苷酸序列：GAAAATTTATATTTTCAAGGGCTACCCGGAACGCCAGGTCCAACTGGTCCGGCGGGCCAAAAAGGCGAGCCGGGGTCGGACGGCATCCCGGGTAGCGCAGGCGAAAAAGGTGAACCGGGCCTGCCGGGCCGTGGCTTCCCGGGCTTCCCGGGAGCGAAAGGCGATAAAGGCAGCAAGGGCGAGGTGGGTTTTCCGGGATTGGCTGGTAGCCCGGGGATTCCGGGCTCCAAGGGCGAGCAGGGTTTTATGGGTCTGCCGGGAACCCCAGGTCCGACCGGTCCGGCGGGTCAGAAAGGTGAACCGGGTAGCGACGGCATTCCGGGCAGCGCGGGTGAAAAAGGTGAACCGGGTTTGCCGGGTCGCGGTTTTCCGGGTTTCCCGGGTGCTAAAGGCGACAAAGGTTCCAAGGGTGAAGTTGGTTTTCCGGGACTGGCGGGCAGCCCGGGCATCCCAGGCTCCAAGGGCGAACAAGGCTTTATGGGTCTGCCGGGCACCCCGGGTCCGACCGGTCCTGCGGGTCAAAAGGGTGAGCCGGGTAGCGATGGTATTCCGGGCTCTGCTGGCGAGAAAGGTGAGCCGGGCCTGCCGGGCCGTGGTTTCCCGGGATTCCCTGGTGCCAAGGGTGACAAGGGTTCGAAGGGCGAGGTTGGTTTTCCGGGCCTGGCGGGTTCTCCGGGTATCCCGGGTAGCAAGGGGGAGCAGGGTTTTATGGGCTTACCGGGAACCCCGGGCCCAACGGGTCCGGCAGGTCAGAAAGGGGAACCGGGCAGCGATGGTATCCCGGGCAGTGCCGGTGAGAAGGGCGAGCCGGGCCTGCCCGGCCGTGGTTTCCCTGGTTTCCCGGGTGCAAAAGGTGATAAAGGTTCCAAGGGCGAAGTGGGTTTCCCGGGTCTCGCGGGCAGCCCGGGCATTCCGGGGTCTAAGGGTGAGCAGGGTTTCATG(SEQID NO.6)。
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(硫酸卡那霉素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(终浓度为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(样品名称记作“菌液”)。
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，完成后收集菌液并进行电泳检测(样品名称分别记作“均一”、“均二”或“超声”)。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，取上清液和沉淀进行电泳检测(样品名称分别记作“上清”和“沉淀”)。
6.将重组Ⅳ型人源化胶原蛋白进行纯化和酶切，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，取流穿进行电泳检验(样品名称记作“流穿”)。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，并取洗杂流穿进行电泳检验(样品名称记作“洗杂”)。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(样品名称记作“洗脱”)。e.用1M咪唑工作液清洗柱材，流速为10mL/min，对流穿进行电泳检测(样品名称记作“1M洗”)。
(2)酶切：按蛋白总质量与TEV酶总质量比为20：1，添加TEV酶，16℃酶切2h，取酶切前和酶切后的样品进行电泳检测(样品名称分别记作“切前”和“切后”)。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，取蛋白液样品进行电泳检测(样品名称记作“换液后”)。
(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，并进行电泳检测(样品名称记作“FL”)。c.梯度洗脱：分别设置0％-15％B液水溶液2min然后保持3个CV、15％-30％B液水溶液2min然后保持3个CV、30％-50％B液水溶液2min然后保持3个CV、50％-100％B液水溶液2min然后保持3个CV，其中，B液含有1M氯化钠和20mM Tris，上述百分比为体积分数，出峰收集并进行电泳检测(样品名称记作“B洗脱”)。d.清洗柱材。e.检测目的蛋白含量并计算蛋白收率，将蛋白储存在4℃环境中。
(4)换水：将收集的蛋白液加入10kDa的超滤浓缩管，进行3500rpm离心15min浓缩换液重复三次，进行电泳检测(样品名称记作“换水”)。
7.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3，图4为重组Ⅳ型人源化胶原蛋白C4P7C6和C007的表达稳定情况对比。其中：
图1和图4中的部分为重组Ⅳ型人源化胶原蛋白C007电泳检测结果，可见纯化及酶切效果较好。
图2和图4中的部分为重组Ⅳ型人源化胶原蛋白C4P7C6电泳检测结果，可见纯化及酶切效果较好。
图3为重组Ⅳ型人源化胶原蛋白C4P7E5电泳检测结果，可见纯化及酶切效果较好。
实施例2：重组Ⅳ型人源化胶原蛋白的生物活性检测
胶原蛋白的活性检测方法可以参考文献Juming Yao,Satoshi Yanagisawa,Tetsuo Asakura,Design,Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens,J Biochem.136,643-649(2004)。具体实施方法如下：
(1)利用紫外吸收法检测待测蛋白样品的浓度，包括商品化的人胶原蛋白(Sigma，C7774)(作为阳性对照)、本发明提供的纯化及酶切效果较好的重组Ⅳ型人源化胶原蛋白C4P7C6、C4P7E5、C007。
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：细胞贴壁率＝(测试孔-空白孔)/(阳性孔-空白孔)×100％。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5所示，从对比中可知，相比于商品化的人胶原蛋白，本发明的重组Ⅳ型人源化胶原蛋白具有更加优秀的生物黏附活性，即：重组Ⅳ型人源化胶原蛋白C4P7C6＞C007＞C4P7E5＞人胶原蛋白。
实施例3：重组Ⅳ型人源化胶原蛋白的质谱检测
实验方法：
表1质谱检测仪器参数
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2 C007质谱检出分子量及对应多肽

检出多肽片段的覆盖率为75.08％，检测结果非常可信。
表3 C4P7E5质谱检出分子量及对应多肽
检出多肽片段的覆盖率为100％，检测结果非常可信。
表4 C4P7C6质谱检出分子量及对应多肽
检出多肽片段的覆盖率为100％，检测结果非常可信。

Claims (10)

  1. 一种重组IV型人源化胶原蛋白，其特征在于，所述重组IV型人源化胶原蛋白的氨基酸序列包含n个重复序列，n为大于等于1的整数；其中，所述重复序列为人天然IV型胶原蛋白的功能区序列；
    优选的，所述n为1、2、3、4、5、6、7或8，其中，当n为大于等于2的整数时，各重复序列之间是直接连接的；
    更优选的，所述重组IV型人源化胶原蛋白的氨基酸序列包含如下(i)-(iii)中的任一项：
    (i)如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的氨基酸序列；
    (ii)在如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的氨基酸序列中添加、取代、缺失或修饰1个或多个氨基酸残基并且保留人天然IV型胶原蛋白的细胞黏附效果的氨基酸序列；
    (iii)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留人天然IV型胶原蛋白的细胞黏附效果，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。
  2. 根据权利要求1所述的重组IV型人源化胶原蛋白，其特征在于，所述重组IV型人源化胶原蛋白的氨基酸序列还包含能够通过TEV蛋白酶切除的氨基酸序列，所述能够通过TEV蛋白酶切除的氨基酸序列如SEQ ID NO.7所示；
    优选的，所述能够通过TEV蛋白酶切除的氨基酸序列与所述重复序列直接连接；
    优选的，所述能够通过TEV蛋白酶切除的氨基酸序列位于所述重组IV型人源化胶原蛋白的N末端。
  3. 一种编码权利要求2所述的重组IV型人源化胶原蛋白的多核苷酸；
    优选的，所述多核苷酸的序列包含如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的序列。
  4. 一种重组表达载体，其特征在于，所述重组表达载体包含权利要求3所述的多核苷酸；
    优选的，所述重组表达载体包括pET系列载体、穿梭载体、噬菌体或病毒载体；
    更优选的，所述重组表达载体为pET-28a-Trx-His。
  5. 一种重组宿主细胞，其特征在于，所述重组宿主细胞包含权利要求4所述的重组表达载体；
    优选的，所述重组宿主细胞为原核细胞、酵母或真核细胞；
    更优选的，所述重组宿主细胞为大肠杆菌BL21(DE3)。
  6. 权利要求1或2所述的重组IV型人源化胶原蛋白的制备方法，其包括以下步骤：
    S1：发酵培养根据权利要求5所述的重组宿主细胞，诱导蛋白表达；
    S2：收集所述蛋白；
    S3：纯化所述蛋白；优选的，包括使用Ni亲和层析柱和/或离子交换层析柱纯化所述蛋白，以及任选的对所述蛋白进行酶切，优选用TEV蛋白酶酶切所述蛋白，更优选的，所述蛋白与所述TEV蛋白酶的质量比为(15-25):1。
  7. 根据权利要求6所述的制备方法，其特征在于，在所述S1步骤中，所述发酵培养在转速为200-240rpm、温度为35-38℃的摇床中进行。
  8. 根据权利要求6或7所述的制备方法，其特征在于，在所述S1步骤中，所述诱导蛋白表达的具体程序为：降温至16-30℃并添加IPTG；优选的，所述IPTG的工作浓度为0.3-0.7mM。
  9. 根据权利要求6-8中任一项所述的制备方法，在所述S2步骤中，所述收集所述蛋白的具体程序为：将S1步骤所得到的混合物于4℃环境下5000-7000rpm离心10-15min，利用含有20-30mM Tris、150-250mM氯化钠和15-25mM咪唑的水溶液重悬沉淀，高压均质或超声破碎细胞后于4℃环境下15000-18000rpm离心20-40min。
  10. 权利要求1或2所述的重组IV型人源化胶原蛋白在制备产品中的用途，所述产品包括医疗器械、化妆品、保健品或药物。
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CN113683679A (zh) * 2021-09-15 2021-11-23 山西锦波生物医药股份有限公司 一种重组i型人源化胶原蛋白c1l6t及其制备方法和用途
CN114940712A (zh) * 2022-06-01 2022-08-26 山西锦波生物医药股份有限公司 一种生物合成人体结构性材料的制备方法

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