CN113683681A - Recombinant I-type humanized collagen C1L3T, and preparation method and application thereof - Google Patents

Recombinant I-type humanized collagen C1L3T, and preparation method and application thereof Download PDF

Info

Publication number
CN113683681A
CN113683681A CN202111082095.2A CN202111082095A CN113683681A CN 113683681 A CN113683681 A CN 113683681A CN 202111082095 A CN202111082095 A CN 202111082095A CN 113683681 A CN113683681 A CN 113683681A
Authority
CN
China
Prior art keywords
gly
pro
ala
c1l3t
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111082095.2A
Other languages
Chinese (zh)
Other versions
CN113683681B (en
Inventor
杨霞
武庚风
张永健
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanxi Jinbo Bio Pharmaceutical Co ltd
Original Assignee
Shanxi Jinbo Bio Pharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanxi Jinbo Bio Pharmaceutical Co ltd filed Critical Shanxi Jinbo Bio Pharmaceutical Co ltd
Priority to CN202111082095.2A priority Critical patent/CN113683681B/en
Publication of CN113683681A publication Critical patent/CN113683681A/en
Application granted granted Critical
Publication of CN113683681B publication Critical patent/CN113683681B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Polymers & Plastics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a recombinant I-type humanized collagen C1L3T, a preparation method and application thereof. The recombinant humanized collagen type I C1L3T provided by the invention comprises a sequence shown as SEQ ID No.3, and the recombinant humanized collagen type I C1L3T optionally comprises a sequence shown as SEQ ID No.2, preferably, the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No.3 are directly connected. The amino acid sequence of the recombinant I-type humanized collagen C1L3T prepared by the invention is derived from the natural collagen amino acid sequence, the molecular weight is smaller than that of the natural I-type collagen, and the recombinant I-type humanized collagen has better cell adhesion effect compared with the I-type collagen, and meanwhile, the preparation method is simple, and the collagen with higher yield can be obtained.

Description

Recombinant I-type humanized collagen C1L3T, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a recombinant I-type humanized collagen C1L3T, and a preparation method and application thereof.
Background
Collagen (collagen) is a biopolymer, is a main component in animal connective tissues, is also a functional protein with the largest content and the widest distribution in mammals, and can account for 25% -30% of the total protein, and the content of the collagen in some organisms is even up to more than 80%. Collagen is mainly distributed in the skin, blood vessels, bones, tendons, teeth, cartilage and the like of the human body, is a main matrix and a scaffold of the tissues, protects and binds various tissues, and plays an important physiological function in the body.
The most common structural feature of collagen is the triple-helical structure formed by 3 peptide chains, i.e. the protein is formed by 3 a peptide chains in a right-handed supercoiled manner, and such triple-helical regions are called collagen regions. Each A peptide chain is composed of repeated Gly-X-Y (X, Y represents any amino acid residue except Gly, X is Pro, Y is Hyp) peptide segments on the molecular structure to form a left-hand helix, and 3 chains form a stable triple helix structure by taking the same axis as the center and in a right-hand supercoiling mode under the interaction of the amino acid residues. In organisms, collagen synthesis and modification starts from procollagen, undergoes chemical changes such as hydroxylation, glycosylation and mutual cross-linking, and is complexly regulated by various biological enzymes. Procollagen contains globular heads and tails in addition to collagen chains. Without these heads and tails, the collagen strands do not fold into the correct triple helix, thereby lacking the biological activity of collagen. Therefore, collagen prepared according to the original gene sequence cannot form the correct spatial structure in spontaneous tissues in vitro. Such difficulties have severely hampered the development and production of human collagen.
Collagen products currently on the market are all derived from animal tissues such as pigs, cattle, fish, etc. In terms of the amino acid composition of collagen: the similarity between the mammal pig and the mammal cow is 95 percent, and the similarity between the mammal pig and the mammal cow is 65 percent. Although the similarity of collagen of mammals such as pig and cow to human is high, it is difficult to avoid the risk of viral infection and sensitization. However, the utilization rate of collagen extracted from edible fishes by human bodies is lower than 65%, and the collagen cannot be completely absorbed and utilized by the human bodies.
In summary, the current collagen can only be used in cosmetics and health care products, and cannot exert the original biological function of the collagen at all.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the situations that the collagen prepared according to the original gene sequence in the field is difficult to form a correct spatial structure in the spontaneous tissues in vitro, and heterologous collagen is easy to induce immune response and low in bioavailability, the invention provides recombinant I-type humanized collagen C1L3T, and also provides a preparation method and application thereof.
Means for solving the problems
In a first aspect, the invention provides a recombinant humanized collagen type I C1L3T, wherein the recombinant humanized collagen type I C1L3T comprises a sequence shown as SEQ ID No. 3.
Further, the recombinant type I humanized collagen C1L3T optionally comprises a sequence shown as SEQ ID No. 2; preferably, the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No.3 are directly linked.
Further, the recombinant humanized collagen type I C1L3T described above comprises one or more of the following sequences: an amino acid sequence shown as SEQ ID No. 4; an amino acid sequence which has 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID No.4 and which retains the cell adhesion effect of the amino acid sequence shown as SEQ ID No. 4; an amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID No.4, and which retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4; an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID No.4 and that retains the cell adhesion effect of the amino acid sequence set forth in SEQ ID No.4, said stringent conditions being medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.
In a second aspect, the present invention provides a polynucleotide encoding the recombinant type I humanized collagen C1L3T described above.
In a third aspect, the present invention provides an expression vector comprising the polynucleotide provided in the second aspect above.
In a fourth aspect, the present invention provides a host cell comprising the expression vector provided in the third aspect above.
In a fifth aspect, the present invention provides a method for producing the above recombinant type I humanized collagen C1L3T, comprising the steps of: culturing the host cell provided in the fourth aspect of the present invention described above in a medium and producing a protein; ② harvesting and purifying the protein, preferably purifying the protein with Ni column and/or ion exchange chromatography; (iii) optionally cleaving the protein, preferably with a PPase protease.
In a sixth aspect, the invention provides the use of the above-mentioned recombinant type I humanized collagen C1L3T in the preparation of a product, wherein the product is preferably a tissue engineering product, a cosmetic, a health product or a medicament.
In a seventh aspect, the present invention provides a product comprising the above-described recombinant type I humanized collagen C1L3T, wherein the product is preferably a tissue engineering product, a cosmetic, a nutraceutical or a pharmaceutical.
In an eighth aspect, the invention provides a use of the recombinant humanized collagen type I C1L3T in preparation of a product with a cell adhesion promoting effect.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme, the amino acid sequence of the recombinant humanized collagen I C1L3T prepared by the invention is derived from the amino acid sequence of natural collagen, the molecular weight of the recombinant humanized collagen I is smaller than that of the natural collagen I, the recombinant humanized collagen I has a better cell adhesion effect compared with the collagen I, and meanwhile, the preparation method is simple, and the collagen with higher yield can be obtained.
Drawings
FIG. 1 is a plasmid map of recombinant expression vector pET32a-C1L3T, wherein the corresponding amino acid sequence of C1L3T is SEQ ID No. 4.
FIG. 2 is a gel electrophoresis diagram of protein C1L3T after induction expression and purification, lane 1 is molecular weight Marker, lane 2 is C1L3T protein after PPase digestion, lane 3 is C1L3T protein after Ni column purification, lane 4 is C1L3T protein after Capto Q ion exchange column purification.
FIG. 3 shows the results of cell adhesion activity assays for commercial human collagen and protein C1L 3T.
Detailed Description
The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. Unless otherwise indicated, the instrumentation, reagents, materials, etc. used in the present invention are commercially available in a conventional manner.
In the present invention, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
In the present invention, "tissue engineering product" means a product used for tissue engineering. Tissue engineering is an emerging discipline for the construction of tissues or organs in vitro or in vivo, combining cell biology and material science.
In the present invention, "medical device" refers to instruments, devices, instruments, in-vitro diagnostic reagents and calibrators, materials and other similar or related items that are used directly or indirectly in the human body.
In the present invention, "medium stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. For guidance in performing hybridization reactions see Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either may be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are washed 2 times in 6 x sodium chloride/sodium citrate (SSC), at about 45 ℃, then at least 50 ℃, in 0.2 x SSC, 0.1% SDS (for low stringency conditions, the wash temperature can be raised to 55 ℃); (2) moderate stringency hybridization conditions are 1 or more washes in 6 XSSC, at about 45 ℃, then 60 ℃ in 0.2 XSSC, 0.1% SDS; (3) high stringency hybridization conditions are 1 or more washes in 6 XSSC, at about 45 ℃, then 65 ℃ in 0.2 XSSC, 0.1% SDS and preferably; (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS, 1 or more washes in 0.2 XSSC, 1% SDS at 65 ℃.
In the present invention, the host cell may be a prokaryotic cell or a eukaryotic cell, such as bacteria of the family Enterobacteriaceae, fungi, yeast, and the like. The skilled worker can replace E.coli strains by other expression strains as host cells.
In the present invention, the nucleic acid molecule comprises a nucleic acid sequence encoding the recombinant type I humanized collagen C1L3T of the present invention. The nucleic acid may be DNA or cDNA. The nucleic acid molecule may consist essentially of a nucleic acid sequence encoding a protein according to the invention or may consist of only a nucleic acid sequence encoding a protein according to the invention. Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, nucleic acid molecules of different nucleic acid sequences can encode the same amino acid sequence.
In the present invention, the nucleic acid sequence of the present invention is included in the vector. Suitable vectors are known in the art of vector construction and include promoter selection and other regulatory elements, such as enhancer elements. The vectors of the invention include sequences suitable for introduction into a cell. For example, the vector may be an expression vector in which the coding sequence for the protein is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in a host cell, or the like.
In the present invention, the term "vector" includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Suitable phage and viral vectors include, but are not limited to: lambda-phage, EMBL phage, simian virus, verruca bovis, Epstein-Barr virus, adenovirus, herpes virus, murine sarcoma virus, murine mammary carcinoma virus, lentivirus, and the like.
In the present invention, the recombinant type I humanized collagen C1L3T can be prepared by a conventional method in the art. For example, it can be produced by the following steps: (1) constructing escherichia coli genetic engineering bacteria: a. obtaining a target gene segment; b. inserting the target gene fragment into a pET-32a expression vector to obtain a corresponding recombinant expression plasmid; c. transferring the recombinant expression plasmid into an escherichia coli competent cell BL21(DE3), and screening to obtain the positive escherichia coli genetic engineering bacteria. (2) Fermentation culture of escherichia coli genetic engineering bacteria and induction and expression of protein: a. selecting single colony of the optimized Escherichia coli genetic engineering bacteria, and suspending in LB liquid containing ampicillin for cultureCulturing at 37 deg.C and 220rpm for 5 hr, and cooling to 16 deg.C; b. the induction was carried out by adding IPTG at a final concentration of 0.25mM, and the culture was carried out for 18 hours. The cells were collected by centrifugation at 3000rpm at 4 ℃ for 20 min. (3) Purification and optional cleavage of the protein: a. resuspending the bacteria in Tris buffer (25mM Tris, 200mM NaCl, pH8.0), homogenizing and disrupting, centrifuging at 17000rpm at 4 ℃ for 20 minutes, and collecting the supernatant; b. by using Ni6FF affinity column binding protein, rinsing the hybrid protein with a solution containing 20mM imidazole (20mM imidazole, 25mM Tris, 200mM NaCl, pH8.0), eluting the protein of interest with a solution containing 250mM imidazole (250mM imidazole, 25mM Tris, 200mM NaCl, pH 8.0); c. adding a proper amount of Prescission Protease (PPase) Protease into the eluted protein sample, and performing enzyme digestion for 2h at the temperature of 16 ℃; d. purifying target protein by using a Capto Q ion exchange column, dialyzing the enzyme-cut protein, changing the solution into A solution (20mM Tris, 10mM NaCl, pH8.0), passing through the Capto Q column, and collecting flow-through solution, namely the target protein without carrier protein.
In the invention, the amino acid sequence of the recombinant humanized collagen type I C1L3T is partially derived from human collagen type I. The type I human collagen has the following sequence:
MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGAR GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKG ADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGE PGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL(SEQ ID No.1)。
in some embodiments, the recombinant humanized collagen type I C1L3T of the present invention comprises a sequence shown by SEQ ID No.3, wherein the sequence shown by SEQ ID No.3 is a type I human collagen peptide fragment, i.e. underlined part of SEQ ID No. 1.
GAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEP(SEQ ID No.3)
In some preferred embodiments, the recombinant humanized collagen type I C1L3T of the present invention comprises a sequence shown by SEQ ID No.3 and a sequence shown by SEQ ID No.2 (GAPGPCCGG), wherein the sequence shown by SEQ ID No.2 is a peptide segment for enhancing collagen activity, and preferably, the sequence shown by SEQ ID No.3 and the sequence shown by SEQ ID No.2 are directly connected.
In some more preferred embodiments, the recombinant type I humanized collagen C1L3T of the present invention comprises a sequence in which one or more amino acids are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID No.4 or in the sequence shown in SEQ ID No.4, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4. The "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
GAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGAPGPCCGG(SEQ ID No.4)
In the present invention, amino acid addition refers to the addition of amino acids to the amino acid sequence, for example, to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID No.4, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4.
In the present invention, amino acid substitution means that a certain amino acid residue at a certain position in an amino acid sequence, for example, an amino acid sequence represented by SEQ ID No.4, is substituted with another amino acid residue, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence represented by SEQ ID No. 4.
In the present invention, the amino acid insertion means that an amino acid residue is inserted at an appropriate position in an amino acid sequence, for example, in the amino acid sequence shown in SEQ ID No.4, and the inserted amino acid residues may be adjacent to each other in whole or in part, or none of the inserted amino acids may be adjacent to each other, as long as the recombinant humanized collagen type I C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4.
In the present invention, the amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from the amino acid sequence, for example, from the amino acid sequence shown in SEQ ID No.4, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4.
In the present invention, the substitution may be a conservative amino acid substitution, which means that 3, preferably 2 or 1 amino acids are substituted with amino acids having similar or similar properties to those of the amino acid sequence shown in SEQ ID No.4 to form a peptide. These conservative variant peptides can be generated by amino acid substitutions according to the following table.
Initial residue(s) Representative substitutions Preferred substitutions
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
In practical applications, the protein polypeptide of the present invention or its pharmaceutically acceptable salt, its derivative or its pharmaceutically acceptable salt, the conjugate, the polymer and the composition can be administered directly to a patient as a medicament or can be administered to a patient after being mixed with a suitable carrier or excipient. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Wherein the suppository can be vaginal suppository, vaginal ring, ointment, cream or gel suitable for vaginal application. The protein polypeptide dosage form can be common preparation, sustained release preparation, controlled release preparation and various particle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.
The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, and the like; for buccal administration, e.g., rectally, vaginally, and sublingually; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above route of administration is preferably by injection, and the preferred route of injection is subcutaneous injection.
The administration dose of the protein polypeptide of the present invention or its pharmaceutically acceptable salt, its derivative or its pharmaceutically acceptable salt, the above conjugate, the above multimer and the above composition depends on many factors, such as the nature and severity of the disease to be prevented or treated, sex, age, body weight and individual reaction of the patient or animal, the specific active ingredient used, the administration route and administration frequency, and the like. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms. For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration and rate of excretion of the particular active ingredient employed; the duration of treatment; drugs used in combination or concomitantly with the specific active ingredient employed; and similar factors known in the medical arts. For example, it is common in the art to start doses of the active ingredient at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.
In order to more clearly express the technical scheme of the invention, the following is further described with reference to specific examples, but the invention is not limited to the specific examples, and the specific examples are only a part of the examples of the invention.
Example 1: preparation of recombinant type I humanized collagen C1L3T
1. Construction of C1L3T Gene expression vector
The total length of the amino acid sequence of the recombinant humanized collagen type I C1L3T used in this example is 234aa of the sequence shown in SEQ ID No.4, the corresponding gene has a total length of 702bp, and codon optimization is performed on codons of escherichia coli, and the optimized sequence is: GGAGCAGTAGGACCCGCTGGGAAAGATGGTGAGGCAGGCGCGCAGGGTCCACCGGGCCCGGCTGGCCCGGCTGGCGAACGTGGCGAGCAAGGTCCGGCGGGTTCTCCGGGATTCCAGGGTCTGCCTGGCCCGGCGGGTCCGCCTGGCGAAGCTGGCAAACCGGGCGAGCAAGGCGTTCCGGGGGACCTGGGTGCACCGGGCCCGAGCGGTGCCCGTGGTGAAAGAGGTTTTCCGGGCGAGCGCGGTGTCCAGGGCCCACCGGGGCCGGCGGGTCCGCGTGGTGCGAACGGTGCCCCTGGTAATGATGGTGCGAAAGGTGATGCAGGTGCGCCAGGCGCGCCAGGCTCGCAAGGTGCTCCGGGCTTACAGGGTATGCCGGGCGAGCGCGGTGCGGCAGGCCTGCCGGGGCCCAAGGGCGACCGTGGTGACGCAGGTCCGAAGGGCGCCGATGGTAGCCCGGGTAAAGATGGCGTGCGTGGTTTGACCGGTCCCATCGGTCCGCCGGGCCCGGCGGGCGCGCCGGGCGACAAAGGCGAAAGCGGTCCGTCCGGCCCGGCGGGCCCGACGGGTGCTCGTGGTGCGCCAGGTGACCGCGGTGAACCGGGCCCACCGGGTCCGGCGGGCTTCGCCGGCCCGCCGGGCGCGGATGGTCAGCCGGGTGCCAAGGGTGAGCCGGGTGCACCGGGTCCGTGTTGTGGTGGT (SEQ ID No. 5).
The synthesis of the gene fragment is carried out by Beijing Shengyue George gene biotechnology, Inc., and the synthesized gene fragment is inserted between multiple cloning sites of pET-32a expression vector by utilizing restriction enzymes BamHI and XhoI to obtain a corresponding recombinant expression plasmid pET32a-C1L3T, wherein the detailed plasmid map is shown in figure 1.
2. Transformation of recombinant expression plasmids
The recombinant expression plasmid is transferred into an escherichia coli competent cell BL21(DE3), and positive escherichia coli genetic engineering bacteria are obtained through screening.
The specific process is as follows: adding 1 mu L of the recombinant expression plasmid into 100 mu L of escherichia coli competent cells BL21(DE3), and standing for 30min on ice; secondly, placing the mixture in a water bath kettle at 42 ℃ for 90s through heat shock, and then rapidly placing the mixture on ice for standing for 2 min; ③ adding 700. mu.L of antibiotic-free LB (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride) to the mixture, and culturing at 37 ℃ and 220rpm for 1 hour; mu.L of the bacterial liquid is evenly coated on an LB plate containing ampicillin (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 mu.g/mL ampicillin); fifthly, the plate is inversely cultured in a 37 ℃ incubator for about 16 hours until a clear and visible colony grows out.
3. Inducible expression of a protein of interest
A single colony of the preferred Escherichia coli genetic engineering bacteria is picked up, placed in an LB liquid culture medium containing ampicillin, cultured at 37 ℃ and 220rpm for 5 hours, cooled to 16 ℃, and then added with IPTG with the final concentration of 0.25mM for induction and cultured for 18 hours. The cells were collected by centrifugation at 3000rpm at 4 ℃ for 20 min.
Purification of C1L3T
(1)Ni6FF affinity column purified protein
Resuspending the bacteria in Tris buffer (25mM Tris, 200mM NaCl, pH8.0), homogenizing and disrupting, centrifuging at 17000rpm at 4 ℃ for 20 minutes, and collecting the supernatant; washing the Ni affinity column with clean water, equilibrating the column with buffer 1(25mM Tris, 200mM NaCl, pH8.0), loading, rinsing the hybrid protein with a solution containing 20mM imidazole (20mM imidazole, 25mM Tris, 200mM NaCl, pH8.0), eluting the protein of interest with a solution containing 250mM imidazole (250mM imidazole, 25mM Tris, 200mM NaCl, pH 8.0); the column was washed with a solution containing 1M imidazole, then with water, and finally packed with 20% ethanol.
(2) Enzyme-digested purified protein
The eluted protein sample was digested with His-tagged Prescission Protease (PPase) at 16 ℃ for 2 h.
(3) Protein purification by Capto Q ion exchange column
Dialyzing the digested protein and changing the solution into solution A (20mM Tris, 10mM NaCl, pH 8.0); and (3) balancing a Capto Q (Cytiva company, a cargo number: 17531610) ion exchange column by using 5 times of column volume of the solution A, passing the protein subjected to enzyme digestion liquid exchange through the Capto Q column, and collecting flow-through liquid, namely the target protein without carrier protein.
Electrophoretic detection of C1L3T
The purity of the C1L3T protein obtained above was checked by SDS-PAGE. The specific process is as follows: 20 mu L of purified protein solution is taken, 5 mu L of 5 multiplied protein loading buffer solution (250mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% beta-mercaptoethanol) is added, the mixture is placed in boiling water at 100 ℃ for boiling for 5min, 10 mu L of each hole is added into SDS-PAGE protein gel, after electrophoresis is carried out for 1h at the voltage of 150V, protein staining is carried out for 3min by Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% ethanol, 10% glacial acetic acid), and then protein destaining solution (10% acetic acid, 5% ethanol) is utilized for destaining.
The detection result is shown in fig. 2, and the apparent molecular weight of the protein obtained by electrophoresis of C1L3T is 35kDa, and the molecular weight corresponds to C1L3T, which indicates that the recombinant type I humanized collagen C1L3T is correctly expressed.
Example 2: biological activity detection of recombinant I-type humanized collagen C1L3T
Methods for detecting Collagen activity may be found in Juming Yao, Satoshi Yanagiwa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell additive and Crosslinking Sequences Derived from Native Collagen, J biochem.136,643-649 (2004). The specific implementation method comprises the following steps:
(1) the concentration of a protein sample to be detected is detected by using an ultraviolet absorption method, and the protein sample comprises commercial human collagen (Sigma, C7774) and the recombinant I-type humanized collagen C1L3T provided by the invention.
Specifically, the protein concentration was calculated by using the empirical formula C (μ g/mL) 144 × (a215-a225) to measure the ultraviolet absorption at 215nm and 225nm, respectively, and it was noted that the detection was performed in the case of a215< 1.5. The principle of the method is as follows: the characteristic absorption of peptide bond under far ultraviolet light is measured, the detection is not influenced by chromophore content, interference substances are few, the operation is simple and convenient, and the method is suitable for detecting human collagen and analogues thereof which do not develop color in Coomassie brilliant blue. After the protein concentration was determined, the concentration of all proteins to be tested was adjusted to 0.5mg/ml with PBS.
(2) 100 μ L of each protein solution and a blank PBS solution control were added to a 96-well plate and allowed to stand at room temperature for 60 min.
(3) Adding 10 into each hole53T3 cells in good culture condition were incubated at 37 deg.C60min。
(4) Each well was washed 4 times with PBS.
(5) OD per well was detected with LDH detection kit (Roche, 04744926001)492nm. According to the value of the blank control, the adherence rate of the cells can be calculated. The calculation formula is as follows: cell adherence rate ═ 100%/(positive well-blank well). The anchorage rate of the cells can reflect the activity of the collagen. The higher the activity of the protein, the better the external environment can be provided for the cells in a short time, and the cells are attached to the wall.
As shown in fig. 3, it is understood from comparison that the recombinant type I humanized collagen C1L3T of the present invention has more excellent bioadhesive activity than the commercial human collagen.
Sequence listing
<110> Shanxi brocade biomedical products Ltd
<120> recombinant I-type humanized collagen C1L3T, and preparation method and application thereof
<130> 6C39-2183051I
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1464
<212> PRT
<213> Homo sapiens
<400> 1
Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr
1 5 10 15
Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp
20 25 30
Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His
35 40 45
Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp
50 55 60
Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn
65 70 75 80
Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro
85 90 95
Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly
100 105 110
Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro
115 120 125
Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro
130 135 140
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala
145 150 155 160
Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser
165 170 175
Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro
180 185 190
Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro
195 200 205
Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
210 215 220
Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg
225 230 235 240
Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro
245 250 255
Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly
260 265 270
Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu
275 280 285
Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg
290 295 300
Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly
305 310 315 320
Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro
325 330 335
Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys
340 345 350
Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly
355 360 365
Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro
370 375 380
Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn
385 390 395 400
Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly
405 410 415
Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn
420 425 430
Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys
435 440 445
Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly
450 455 460
Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu
465 470 475 480
Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro
485 490 495
Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly
500 505 510
Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg
515 520 525
Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro
530 535 540
Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly
545 550 555 560
Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln
565 570 575
Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro
580 585 590
Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly
595 600 605
Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro
610 615 620
Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro
625 630 635 640
Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly
645 650 655
Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro
660 665 670
Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln
675 680 685
Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly
690 695 700
Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser
705 710 715 720
Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala
725 730 735
Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly
740 745 750
Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro
755 760 765
Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser
770 775 780
Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly
785 790 795 800
Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro
805 810 815
Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala
820 825 830
Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly
835 840 845
Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala
850 855 860
Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala
865 870 875 880
Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly
885 890 895
Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu
900 905 910
Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro
915 920 925
Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly
930 935 940
Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val
945 950 955 960
Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro
965 970 975
Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly
980 985 990
Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro
995 1000 1005
Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala Glu Gly Ser Pro
1010 1015 1020
Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly
1025 1030 1035 1040
Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro
1045 1050 1055
Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala
1060 1065 1070
Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly
1075 1080 1085
Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp
1090 1095 1100
Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro
1105 1110 1115 1120
Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly
1125 1130 1135
Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys
1140 1145 1150
Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg
1155 1160 1165
Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly
1170 1175 1180
Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe
1185 1190 1195 1200
Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr
1205 1210 1215
Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp
1220 1225 1230
Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro
1235 1240 1245
Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met
1250 1255 1260
Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln
1265 1270 1275 1280
Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly
1285 1290 1295
Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp
1300 1305 1310
Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu
1315 1320 1325
Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp
1330 1335 1340
Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr
1345 1350 1355 1360
Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr
1365 1370 1375
Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly
1380 1385 1390
Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr
1395 1400 1405
Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys
1410 1415 1420
Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile
1425 1430 1435 1440
Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe
1445 1450 1455
Asp Val Gly Pro Val Cys Phe Leu
1460
<210> 2
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly Ala Pro Gly Pro Cys Cys Gly Gly
1 5
<210> 3
<211> 225
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly
1 5 10 15
Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro
20 25 30
Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro
35 40 45
Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly
50 55 60
Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu
65 70 75 80
Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn
85 90 95
Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly
100 105 110
Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu
115 120 125
Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala
130 135 140
Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly
145 150 155 160
Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp
165 170 175
Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg
180 185 190
Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly
195 200 205
Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu
210 215 220
Pro
225
<210> 4
<211> 234
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly
1 5 10 15
Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro
20 25 30
Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro
35 40 45
Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly
50 55 60
Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu
65 70 75 80
Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn
85 90 95
Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly
100 105 110
Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu
115 120 125
Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala
130 135 140
Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly
145 150 155 160
Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp
165 170 175
Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg
180 185 190
Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly
195 200 205
Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu
210 215 220
Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly
225 230
<210> 5
<211> 702
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggagcagtag gacccgctgg gaaagatggt gaggcaggcg cgcagggtcc accgggcccg 60
gctggcccgg ctggcgaacg tggcgagcaa ggtccggcgg gttctccggg attccagggt 120
ctgcctggcc cggcgggtcc gcctggcgaa gctggcaaac cgggcgagca aggcgttccg 180
ggggacctgg gtgcaccggg cccgagcggt gcccgtggtg aaagaggttt tccgggcgag 240
cgcggtgtcc agggcccacc ggggccggcg ggtccgcgtg gtgcgaacgg tgcccctggt 300
aatgatggtg cgaaaggtga tgcaggtgcg ccaggcgcgc caggctcgca aggtgctccg 360
ggcttacagg gtatgccggg cgagcgcggt gcggcaggcc tgccggggcc caagggcgac 420
cgtggtgacg caggtccgaa gggcgccgat ggtagcccgg gtaaagatgg cgtgcgtggt 480
ttgaccggtc ccatcggtcc gccgggcccg gcgggcgcgc cgggcgacaa aggcgaaagc 540
ggtccgtccg gcccggcggg cccgacgggt gctcgtggtg cgccaggtga ccgcggtgaa 600
ccgggcccac cgggtccggc gggcttcgcc ggcccgccgg gcgcggatgg tcagccgggt 660
gccaagggtg agccgggtgc accgggtccg tgttgtggtg gt 702

Claims (10)

1. A recombinant humanized collagen type I C1L3T, wherein the recombinant humanized collagen type I C1L3T comprises a sequence shown as SEQ ID No. 3.
2. The recombinant type I humanized collagen C1L3T according to claim 1, wherein said recombinant type I humanized collagen C1L3T optionally comprises the sequence shown as SEQ ID No. 2; preferably, the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No.3 are directly linked.
3. The recombinant humanized collagen type I C1L3T according to claim 1 or 2, characterized in that said recombinant humanized collagen type I C1L3T comprises one or more of the following sequences:
an amino acid sequence shown as SEQ ID No. 4;
an amino acid sequence which has 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID No.4 and which retains the cell adhesion effect of the amino acid sequence shown as SEQ ID No. 4;
an amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID No.4, and which retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4;
an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID No.4 and that retains the cell adhesion effect of the amino acid sequence set forth in SEQ ID No.4, said stringent conditions being medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.
4. A polynucleotide encoding the recombinant type I humanized collagen C1L3T of any one of claims 1 to 3.
5. An expression vector comprising the polynucleotide of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method of producing the recombinant humanized type I collagen C1L3T according to any one of claims 1 to 3, comprising the steps of:
culturing the host cell according to claim 6 in a medium and producing a protein;
② harvesting and purifying the protein, preferably purifying the protein with Ni column and/or ion exchange chromatography;
(iii) optionally cleaving the protein, preferably with a PPase protease.
8. Use of the recombinant humanized collagen type I C1L3T as defined in any one of claims 1 to 3 for the preparation of a product, preferably a tissue engineering product, a cosmetic product, a nutraceutical product or a pharmaceutical product.
9. A product comprising the recombinant humanized collagen type I C1L3T of any of claims 1 to 3, wherein said product is preferably a tissue engineering product, a cosmetic product, a nutraceutical product or a pharmaceutical product.
10. Use of the recombinant humanized collagen type I C1L3T as claimed in any one of claims 1 to 3 for the preparation of a product having a cell adhesion promoting effect.
CN202111082095.2A 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L3T and preparation method and application thereof Active CN113683681B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111082095.2A CN113683681B (en) 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L3T and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111082095.2A CN113683681B (en) 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L3T and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN113683681A true CN113683681A (en) 2021-11-23
CN113683681B CN113683681B (en) 2023-10-03

Family

ID=78586414

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111082095.2A Active CN113683681B (en) 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L3T and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN113683681B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114940712A (en) * 2022-06-01 2022-08-26 山西锦波生物医药股份有限公司 Preparation method of biosynthesized human structural material

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103122027A (en) * 2012-11-26 2013-05-29 杨霞 Recombinant human collagen and production method thereof
AU2016203028A1 (en) * 2013-03-08 2016-05-26 Novartis Ag Peptides and compositions for treatment of joint damage
CN106798696A (en) * 2017-03-02 2017-06-06 山西锦波生物医药股份有限公司 A kind of biological toothpaste for nursing and its production method containing recombination human source collagen
CN107857812A (en) * 2017-11-17 2018-03-30 杭州惠博士生物科技有限公司 A kind of preparation method of same human-like collagen amino acid, gene order and Argine Monohydrochloride
CN109293783A (en) * 2018-10-25 2019-02-01 山西锦波生物医药股份有限公司 Polypeptide, its production method and purposes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103122027A (en) * 2012-11-26 2013-05-29 杨霞 Recombinant human collagen and production method thereof
AU2016203028A1 (en) * 2013-03-08 2016-05-26 Novartis Ag Peptides and compositions for treatment of joint damage
CN106798696A (en) * 2017-03-02 2017-06-06 山西锦波生物医药股份有限公司 A kind of biological toothpaste for nursing and its production method containing recombination human source collagen
CN107857812A (en) * 2017-11-17 2018-03-30 杭州惠博士生物科技有限公司 A kind of preparation method of same human-like collagen amino acid, gene order and Argine Monohydrochloride
CN109293783A (en) * 2018-10-25 2019-02-01 山西锦波生物医药股份有限公司 Polypeptide, its production method and purposes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
崔福斋: "新型促细胞粘附重组人源性胶原的原核表达及功能性研究", 中国生物工程杂志, vol. 27, no. 8, pages 1 - 6 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114940712A (en) * 2022-06-01 2022-08-26 山西锦波生物医药股份有限公司 Preparation method of biosynthesized human structural material
CN114940712B (en) * 2022-06-01 2023-12-26 山西锦波生物医药股份有限公司 Preparation method of biological synthetic human body structural material

Also Published As

Publication number Publication date
CN113683681B (en) 2023-10-03

Similar Documents

Publication Publication Date Title
CN113621052B (en) Recombinant I-type humanized collagen polypeptide and preparation method and application thereof
CN113683680B (en) Recombinant I-type humanized collagen C1L1T and preparation method and application thereof
CN113683679B (en) Recombinant I-type humanized collagen C1L6T and preparation method and application thereof
CN113637068B (en) Recombinant I-type humanized collagen C1L5T and preparation method and application thereof
CN110845603B (en) Human collagen 17-type polypeptide, production method and use thereof
CN111944057B (en) Recombinant human collagen peptide and application thereof
CN109575126B (en) Polypeptides, method for the production and use thereof
JP2740417B2 (en) Preparation method of human nerve growth factor by genetic recombination
CN114805551B (en) Recombinant type III collagen and preparation method thereof
JPH04502916A (en) Purified ciliary neurotrophic factor
JP2002526073A (en) A coding sequence for a novel human growth differentiation factor, a polypeptide encoded by the DNA sequence thereof, and a method for producing them.
CN110066342B (en) Hybrid peptide with functions of immunoregulation, endotoxin neutralization and digestion and anti-inflammation, and preparation method and application thereof
CN118047857A (en) Preparation method of biological synthetic human body structural material
CN110128544B (en) Hybrid peptide with immunoregulation and anti-inflammatory functions and preparation method and application thereof
CN113683681B (en) Recombinant I-type humanized collagen C1L3T and preparation method and application thereof
KR100861240B1 (en) A method of producing biologically active human acidic fibroblst growth factor and its use in promoting angiogenesis
CN113735959B (en) FGF analogue for treating NASH
CN113788891B (en) Recombinant I-type humanized collagen C1L4T and preparation method and application thereof
CN113683678B (en) Recombinant I-type humanized collagen C1L2T and preparation method and application thereof
US7973010B2 (en) Full length polynucleotide coding chicken type II collagen and the use of it
CN117986355B (en) Recombinant humanized III type collagen and preparation method and application thereof
WO2024119724A1 (en) Collagen peptide, preparation method therefor and use thereof
CN114805544B (en) Insulin lispro precursor, recombinant genetic engineering bacterium thereof and construction method thereof
CN114480320B (en) Recombinant night monkey uricase and application thereof
CN118085064A (en) Recombinant polypeptide and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant