CN113788891A - Recombinant I-type humanized collagen C1L4T and preparation method and application thereof - Google Patents

Recombinant I-type humanized collagen C1L4T and preparation method and application thereof Download PDF

Info

Publication number
CN113788891A
CN113788891A CN202111096219.2A CN202111096219A CN113788891A CN 113788891 A CN113788891 A CN 113788891A CN 202111096219 A CN202111096219 A CN 202111096219A CN 113788891 A CN113788891 A CN 113788891A
Authority
CN
China
Prior art keywords
gly
pro
ala
c1l4t
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
CN202111096219.2A
Other languages
Chinese (zh)
Other versions
CN113788891B (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 CN202111096219.2A priority Critical patent/CN113788891B/en
Publication of CN113788891A publication Critical patent/CN113788891A/en
Application granted granted Critical
Publication of CN113788891B publication Critical patent/CN113788891B/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)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Nutrition Science (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Birds (AREA)
  • Physics & Mathematics (AREA)
  • Dermatology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a recombinant I-type humanized collagen C1L4T, a preparation method and application thereof. The recombinant humanized collagen type I C1L4T provided by the invention comprises a sequence shown as SEQ ID No.3, and the recombinant humanized collagen type I C1L4T 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. Compared with the type I collagen, the recombinant I humanized collagen C1L4T prepared by the invention has smaller molecular weight, and better cell adhesion effect, in addition, part of the amino acid sequence of the recombinant I humanized collagen is derived from the amino acid sequence of natural collagen, the biocompatibility is better, the preparation method is simple, and the collagen with higher yield can be obtained at low cost.

Description

Recombinant I-type humanized collagen C1L4T 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 C1L4T, and a preparation method and application thereof.
Background
Collagen (collagen), generally white, transparent, unbranched fibrils, is the basic support for skin and bone, the most abundant class of proteins in the mammalian body, an important constituent of the extracellular matrix. The collagen content in human body is 25% -30% of total protein, and is an important structural protein, and plays an important role in protecting organism and supporting organs.
The types of collagen are many, and the common types are type I, type II, type III, type V and type XI. In normal skin tissue, collagen exists primarily as type I, III collagen fibers. Type I collagen and type iii collagen are closely related to the process and quality of skin injury repair.
At present, collagen mainly has two approaches of animal tissue extraction and gene engineering method preparation. The naturally extracted collagen is a mixture consisting of a plurality of collagens with different molecular weights, is insoluble in water and has poor biocompatibility; furthermore, since it is derived from animal tissues, there is a risk of cross-infection with diseases of animal origin or infectious diseases of human. The collagen prepared by the genetic engineering method can solve the disease infection risk caused by the collagen extracted by the traditional method, and can improve the hydrophilicity and the stability of the collagen and optimize the molecular weight of the collagen by optimizing the amino acid sequence of the collagen; therefore, the prepared collagen has good histocompatibility, skin permeability and safety.
However, the collagen structure of the original gene sequence is complicated and it is affected and regulated by various factors during the synthesis process, so that the collagen prepared according to the original gene sequence cannot form a correct spatial structure in spontaneous tissues in vitro. Such difficulties have severely hampered the development and production of human collagen.
Therefore, in the process of preparing collagen by genetic engineering, it is necessary to select a suitable collagen domain and reasonably optimize the amino acid sequence of the selected collagen domain to improve the preparation efficiency of collagen and the stability and activity of the prepared collagen.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problems that the original gene sequence human collagen is difficult to prepare by a genetic engineering means, the biocompatibility of heterologous collagen is poor and the heterologous collagen is easy to carry pathogens in the field, the invention provides a recombinant I-type humanized collagen C1L4T, and simultaneously 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 C1L4T, wherein the recombinant humanized collagen type I C1L4T comprises a sequence shown as SEQ ID No. 3.
Further, the recombinant type I humanized collagen C1L4T 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 C1L4T described above comprises at least one of the following sequences: an amino acid sequence shown as SEQ ID No. 4; an amino acid sequence having not less than 80% identity to the amino acid sequence represented by SEQ ID No.4 that retains the cell adhesion effect of the amino acid sequence represented by 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, retaining 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 retains the cell adhesion effect of the amino acid sequence shown as SEQ ID No.4, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown as SEQ ID No.4 under stringent conditions, which are medium to very high stringent conditions.
In a second aspect, the present invention provides a polynucleotide encoding the recombinant type I humanized collagen C1L4T 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 C1L4T, comprising the steps of: culturing the host cell provided by the fourth aspect in a culture 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 C1L4T 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 C1L4T, 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 C1L4T in preparation of a product with a cell adhesion promoting effect.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme, compared with the type I collagen, the recombinant humanized collagen C1L4T prepared by the invention has smaller molecular weight and better cell adhesion effect, in addition, part of the amino acid sequence of the recombinant humanized collagen is derived from the amino acid sequence of the natural collagen, the biocompatibility is better, the preparation method is simple, and the collagen with higher yield can be obtained at low cost.
Drawings
FIG. 1 is a map of recombinant expression plasmid pET32a-C1L4T, wherein the corresponding amino acid sequence of C1L4T is SEQ ID No. 4.
FIG. 2 is a gel electrophoresis diagram of protein C1L4T after induction expression and purification, wherein lane 1 is molecular weight Marker, lane 2 is C1L4T protein purified by Ni column, lane 3 is C1L4T protein digested by PPase.
FIG. 3 shows the results of cell adhesion activity assays for commercial human collagen and protein C1L 4T.
Detailed Description
The following describes embodiments of the present invention, but the present invention is not limited to these embodiments.
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 the present invention, "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, "identity" refers to the ratio of identical bases or amino acids between a detected sequence and a target sequence to the entire sequence. Similarity in amino acid sequence alignment also includes whether, in addition to identical residues, two residues at corresponding positions have similar properties, such as size of side chain groups, charge, hydrophilicity and hydrophobicity, and the like.
In the present invention, the amino acid "addition" refers to the addition of an amino acid at the C-terminus or N-terminus of an amino acid sequence, as long as the protein of the present invention retains the cell adhesion effect of the original amino acid sequence.
In the present invention, the amino acid "deletion" means that 1, 2 or 3 or more amino acids may be deleted from the amino acid sequence as long as the protein of the present invention retains the cell adhesion effect of the original amino acid sequence.
In the present invention, the term "insertion" of amino acids means insertion of amino acid residues at appropriate positions in the amino acid sequence, 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 protein of the present invention retains the cell adhesion effect of the original amino acid sequence.
In the present invention, amino acid "substitution" means that a certain amino acid residue at a certain position in an amino acid sequence is substituted with other amino acid residues as long as the protein of the present invention retains the cell adhesion effect of the original amino acid sequence; the term "substitution" may refer to conservative amino acid substitution, which means that 3, preferably 2 or 1 amino acids are substituted by amino acids having similar or similar properties to the original amino acid sequence 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 the present invention, "medium to very high stringency conditions" include "medium stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions", which 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 nucleic acid molecule comprises a nucleic acid sequence encoding the recombinant type I humanized collagen C1L4T 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 host cell may be a prokaryotic cell, such as a bacterium of the family Enterobacteriaceae, or a eukaryotic cell, such as a fungus and a yeast. The skilled worker can replace E.coli strains by other expression strains as host cells.
In the present invention, the recombinant type I humanized collagen C1L4T 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 obtained target gene fragment into a pET-32a expression vector to obtain a 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 a single colony of the optimized escherichia coli genetic engineering bacteria, placing the single colony in a liquid culture medium containing ampicillin, culturing at 37 ℃ and 220rpm for 5 hours, and cooling to 16 ℃; b. induction was carried out by adding IPTG at a final concentration of 0.25mM, and culturing was carried out for 18 hours. The cells were collected by centrifugation at 3000rpm for 20min at 4 ℃.
(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 washing buffer 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. the eluted protein sample was digested with a suitable amount of His-tagged Prescission Protease (PPase) Protease at 16 ℃ for 2 h.
In the invention, a partial sequence in the recombinant humanized collagen type I C1L4T is derived from human type I collagen. The human type I collagen sequence is as follows: MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEP
Figure BDA0003264421660000061
Figure BDA0003264421660000062
Figure BDA0003264421660000063
GPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL(SEQ ID No.1)。
In one embodiment, the recombinant humanized collagen type I C1L4T of the present invention comprises a sequence shown by SEQ ID No.3, wherein the sequence shown by SEQ ID No.3 is a human collagen type I peptide, i.e., the bold underlined portion of the above sequence.
GDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPA(SEQ ID No.3)
In a preferred embodiment, the recombinant humanized collagen type I C1L4T of the present invention comprises a sequence shown as SEQ ID No.3 and a sequence shown as SEQ ID No.2 (GAPGPCCGG), wherein the sequence shown as SEQ ID No.2 is a terminal peptide segment for enhancing collagen activity, and the sequence shown as SEQ ID No.3 and the sequence shown as SEQ ID No.2 are directly connected.
In a more preferred embodiment, the recombinant humanized collagen type I C1L4T of the present invention comprises an amino acid sequence shown as SEQ ID No.4, wherein the amino acid sequence shown as SEQ ID No.4 is formed by combining an amino acid sequence shown as SEQ ID No.3 and an amino acid sequence shown as SEQ ID No.2, the amino acid sequence shown as SEQ ID No.3 is positioned at the N-terminal of the amino acid sequence shown as SEQ ID No.2, and the two are directly connected.
GDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGAPGPCCGG(SEQ ID No.4)
In another more preferred embodiment, the recombinant type I humanized collagen C1L4T of the present invention comprises a sequence in which one or more amino acids are substituted, deleted, inserted and/or added in the sequence shown in SEQ ID No.4, as long as the recombinant type I humanized collagen C1L4T 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.
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.
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention. Unless otherwise indicated, the instrumentation, reagents, materials, etc. used in the present invention are commercially available in a conventional manner.
Example 1: preparation of recombinant type I humanized collagen C1L4T
1. Construction of C1L4T Gene expression vector
The total length of the amino acid sequence of the recombinant humanized collagen type I C1L4T used in this example is a sequence shown by SEQ ID No.4, 222aa in total, the corresponding gene has a total length of 666bp, codon optimization is performed on codons of escherichia coli, and the optimized sequence is:
GGGGATGCGGGAGCAAAAGGTGACGCTGGACCGCCGGGCCCAGCTGGCCCGGCGGGTCCGCCGGGTCCAATTGGTAACGTGGGTGCCCCTGGCGCGAAAGGTGCGCGTGGTAGCGCAGGTCCGCCGGGTGCTACCGGTTTCCCGGGCGCGGCAGGACGTGTGGGCCCGCCGGGCCCGTCCGGTAATGCAGGCCCGCCAGGCCCGCCTGGTCCGGCGGGTAAAGAAGGCGGTAAGGGTCCGCGTGGCGAAACCGGCCCGGCTGGCCGCCCTGGCGAAGTTGGTCCGCCGGGCCCGCCGGGTCCGGCCGGCGAGAAAGGCAGCCCGGGTGCGGACGGTCCGGCGGGTGCGCCAGGAACCCCGGGTCCGCAGGGCATCGCCGGCCAACGTGGAGTTGTCGGTCTGCCGGGCCAAAGAGGTGAGCGCGGTTTTCCGGGCTTGCCGGGTCCATCGGGCGAGCCGGGCAAGCAGGGTCCGAGCGGTGCGAGCGGTGAGCGTGGTCCTCCGGGTCCGATGGGTCCGCCGGGTCTGGCAGGCCCACCGGGTGAGAGCGGTCGTGAAGGTGCCCCAGGCGCGGAAGGCTCTCCGGGCCGCGACGGCTCCCCGGGCGCGAAGGGCGATCGTGGTGAGACGGGTCCGGCTGGTGCACCGGGTCCGTGTTGTGGTGGT(SEQ ID No.5)。
the gene fragment was synthesized by Beijing Shengyue George Gene Biotechnology Co., Ltd, and the synthesized gene fragment was inserted between enzyme cleavage sites BamHI and XhoI of pET-32a expression vector to construct recombinant expression plasmid pET32a-C1L4T, the detailed drawing of which is shown in FIG. 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 plasmid into 100 mu L of escherichia coli competent cells BL21(DE3), uniformly mixing, and standing for 30min on ice; ② the mixture is heated for 90s under the condition of 42 ℃, and then is rapidly placed on ice for standing for 2 min; ③ adding 700 microliter of liquid LB without antibiotics (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride) preheated at 37 ℃ to the mixture, and culturing at 37 ℃ and 220rpm for 1 h; 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, and then clearly visible colonies grow.
3. Inducible expression of a protein of interest
Selecting a single colony of the optimized Escherichia coli genetic engineering bacteria from the plate, placing the single colony in a liquid LB culture medium containing ampicillin, placing the single colony in a constant-temperature shaking table with the rotation speed of 220rpm at 37 ℃ for culturing for 5 hours, cooling to 16 ℃, adding IPTG with the final concentration of 0.25mM for induction, and culturing for 18 hours. The cells were collected by centrifugation at 3000rpm at 4 ℃ for 20 min.
Purification of C1L4T
Resuspending the bacteria in Tris buffer (25mM Tris, 200mM NaCl, pH8.0), lysing, homogenizing, disrupting, centrifuging at 17000rpm at 4 deg.C for 20min, 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 wash buffer 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.
To the eluted protein sample, an appropriate amount of Prescission Protease (PPase) Protease with His tag was added and digested at 16 ℃ for 2 hours.
Electrophoretic detection of C1L4T
The purity of the recombinant humanized collagen type I C1L4T was checked by SDS-PAGE. The specific process is as follows: taking 20 mu L of purified protein solution, adding a proper amount of 5 Xprotein loading buffer solution (250mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% beta-mercaptoethanol) to make the final concentration 1X, placing in 100 ℃ boiling water for 5min, adding 10 mu L of purified protein solution into SDS-PAGE protein gel, performing electrophoresis at 150V for 1h, performing protein staining for 3min by using Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% ethanol, 10% glacial acetic acid), and performing decolorization by using protein decolorizing solution (10% acetic acid, 5% ethanol).
The detection result is shown in fig. 2, and the apparent molecular weight of the protein obtained by electrophoresis of C1L4T is 33kDa, and the molecular weight corresponds to C1L4T, which indicates that the recombinant type I humanized collagen protein C1L4T is correctly expressed.
Example 2: biological activity detection of recombinant I-type humanized collagen C1L4T
(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 C1L4T 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. 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 state were incubated at 37 ℃ for 60 min.
(4) Each well was washed 4 times with PBS.
(5) Absorbance OD at 492nm was detected with LDH detection kit (Roche, 04744926001)492nm. From the values of the blank control, the adherence rate of the cells can be calculated as (test well-blank well) × 100%/(positive well-blank well).
As shown in FIG. 3, it is evident from comparison that the OD of the well to which the recombinant type I humanized collagen C1L4T of the present invention was added was larger than that of the commercially available human collagen492nmLarger means that the anchorage rate of the cells in the well is greater. The protein C1L4T of the invention is proved to provide a good-quality external environment for cells in a shorter time, help the cells to adhere to the wall and have higher bioactivity than commercial human collagen.
Sequence listing
<110> Shanxi brocade biomedical products Ltd
<120> recombinant I-type humanized collagen C1L4T, and preparation method and application thereof
<130> 6C39-2183052I
<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> 213
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly
1 5 10 15
Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala
20 25 30
Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro
35 40 45
Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly
50 55 60
Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro
65 70 75 80
Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro
85 90 95
Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly
100 105 110
Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln
115 120 125
Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro
130 135 140
Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly
145 150 155 160
Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu
165 170 175
Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala Glu
180 185 190
Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly
195 200 205
Glu Thr Gly Pro Ala
210
<210> 4
<211> 222
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly
1 5 10 15
Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala
20 25 30
Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro
35 40 45
Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly
50 55 60
Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro
65 70 75 80
Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro
85 90 95
Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly
100 105 110
Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln
115 120 125
Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro
130 135 140
Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly
145 150 155 160
Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu
165 170 175
Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala Glu
180 185 190
Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly
195 200 205
Glu Thr Gly Pro Ala Gly Ala Pro Gly Pro Cys Cys Gly Gly
210 215 220
<210> 5
<211> 666
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggggatgcgg gagcaaaagg tgacgctgga ccgccgggcc cagctggccc ggcgggtccg 60
ccgggtccaa ttggtaacgt gggtgcccct ggcgcgaaag gtgcgcgtgg tagcgcaggt 120
ccgccgggtg ctaccggttt cccgggcgcg gcaggacgtg tgggcccgcc gggcccgtcc 180
ggtaatgcag gcccgccagg cccgcctggt ccggcgggta aagaaggcgg taagggtccg 240
cgtggcgaaa ccggcccggc tggccgccct ggcgaagttg gtccgccggg cccgccgggt 300
ccggccggcg agaaaggcag cccgggtgcg gacggtccgg cgggtgcgcc aggaaccccg 360
ggtccgcagg gcatcgccgg ccaacgtgga gttgtcggtc tgccgggcca aagaggtgag 420
cgcggttttc cgggcttgcc gggtccatcg ggcgagccgg gcaagcaggg tccgagcggt 480
gcgagcggtg agcgtggtcc tccgggtccg atgggtccgc cgggtctggc aggcccaccg 540
ggtgagagcg gtcgtgaagg tgccccaggc gcggaaggct ctccgggccg cgacggctcc 600
ccgggcgcga agggcgatcg tggtgagacg ggtccggctg gtgcaccggg tccgtgttgt 660
ggtggt 666

Claims (10)

1. A recombinant humanized collagen type I C1L4T, wherein the recombinant humanized collagen type I C1L4T comprises a sequence shown as SEQ ID No. 3.
2. The recombinant type I humanized collagen C1L4T according to claim 1, wherein said recombinant type I humanized collagen C1L4T 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 C1L4T according to claim 1 or 2, characterized in that said recombinant humanized collagen type I C1L4T comprises at least one of the following sequences:
an amino acid sequence shown as SEQ ID No. 4;
an amino acid sequence having not less than 80% identity to the amino acid sequence represented by SEQ ID No.4 that retains the cell adhesion effect of the amino acid sequence represented by 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, retaining 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 retains the cell adhesion effect of the amino acid sequence shown as SEQ ID No.4, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown as SEQ ID No.4 under stringent conditions, which are medium to very high stringent conditions.
4. A polynucleotide encoding the recombinant type I humanized collagen C1L4T according to 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 for producing recombinant humanized type I collagen C1L4T according to any of claims 1 to 3, comprising the following steps:
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 C1L4T according to any 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 C1L4T according to 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 C1L4T as defined in any one of claims 1 to 3 in the preparation of a product having the effect of promoting cell adhesion.
CN202111096219.2A 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L4T and preparation method and application thereof Active CN113788891B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111096219.2A CN113788891B (en) 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L4T and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111096219.2A CN113788891B (en) 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L4T and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN113788891A true CN113788891A (en) 2021-12-14
CN113788891B CN113788891B (en) 2023-10-31

Family

ID=78878903

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111096219.2A Active CN113788891B (en) 2021-09-15 2021-09-15 Recombinant I-type humanized collagen C1L4T and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN113788891B (en)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049159A1 (en) * 1999-02-19 2000-08-24 Terumo Kabushiki Kaisha Collagen-binding physiologically active polypeptide
WO2004085473A2 (en) * 2003-03-28 2004-10-07 Fuji Photo Film B.V. Rgd-enriched gelatine-like proteins with enhanced cell binding
EP1584630A1 (en) * 2004-04-08 2005-10-12 Nederlandse Organisatie voor toegepast-natuurwetenschappelijk Onderzoek TNO Method for preparing a modified protein outside a host cell
CN101148479A (en) * 2007-09-11 2008-03-26 浙江理工大学 Recombination human collagen and biological synthesis method thereof
CN109022464A (en) * 2018-07-02 2018-12-18 西安巨子生物基因技术股份有限公司 The hydroxylacion method of recombination human source collagen type
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
CN110003324A (en) * 2019-03-19 2019-07-12 江苏悦智生物医药有限公司 Recombination human source collagen and its application
CN110903383A (en) * 2019-11-21 2020-03-24 郭伟 Recombinant human type I collagen, encoding gene, engineering bacterium and application thereof
CN111334512A (en) * 2019-12-06 2020-06-26 肽源（广州）生物科技有限公司 Recombinant human-like collagen containing hydroxyproline and hydroxylysine and production method thereof
CN111363028A (en) * 2018-12-25 2020-07-03 江苏江山聚源生物技术有限公司 Recombinant human type I collagen, expression strain and construction method thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049159A1 (en) * 1999-02-19 2000-08-24 Terumo Kabushiki Kaisha Collagen-binding physiologically active polypeptide
WO2004085473A2 (en) * 2003-03-28 2004-10-07 Fuji Photo Film B.V. Rgd-enriched gelatine-like proteins with enhanced cell binding
EP1584630A1 (en) * 2004-04-08 2005-10-12 Nederlandse Organisatie voor toegepast-natuurwetenschappelijk Onderzoek TNO Method for preparing a modified protein outside a host cell
CN101148479A (en) * 2007-09-11 2008-03-26 浙江理工大学 Recombination human collagen and biological synthesis method thereof
CN109022464A (en) * 2018-07-02 2018-12-18 西安巨子生物基因技术股份有限公司 The hydroxylacion method of recombination human source collagen type
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
CN111363028A (en) * 2018-12-25 2020-07-03 江苏江山聚源生物技术有限公司 Recombinant human type I collagen, expression strain and construction method thereof
CN110003324A (en) * 2019-03-19 2019-07-12 江苏悦智生物医药有限公司 Recombination human source collagen and its application
CN110903383A (en) * 2019-11-21 2020-03-24 郭伟 Recombinant human type I collagen, encoding gene, engineering bacterium and application thereof
CN111334512A (en) * 2019-12-06 2020-06-26 肽源（广州）生物科技有限公司 Recombinant human-like collagen containing hydroxyproline and hydroxylysine and production method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENBANK:EAW94630.1: "GENBANK:EAW94630.1", NCBI, pages 1 - 2 *
HÉCTOR CAPELLA-MONSONÍS等: "An experimental toolbox for characterization of mammalian collagen type I in biological specimens", NATURE PROTOCOLS, vol. 13, pages 507 - 529 *
崔福斋等: "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 *

Also Published As

Publication number Publication date
CN113788891B (en) 2023-10-31

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
CN110845603B (en) Human collagen 17-type polypeptide, production method and use thereof
CN113637068B (en) Recombinant I-type humanized collagen C1L5T and preparation method and application thereof
CN111944057B (en) Recombinant human collagen peptide and application thereof
CN109575126B (en) Polypeptides, method for the production and use thereof
CN114805551B (en) Recombinant type III collagen and preparation method thereof
CN112745385A (en) Recombinant humanized collagen, industrial preparation method and product application thereof
CN118047857A (en) Preparation method of biological synthetic human body structural material
CN110066342B (en) Hybrid peptide with functions of immunoregulation, endotoxin neutralization and digestion and anti-inflammation, and preparation method and application thereof
CN112851791B (en) Novel FGF analogue for resisting metabolic disorder and application thereof
CN113683681B (en) Recombinant I-type humanized collagen C1L3T and preparation method and application thereof
CN113788891B (en) Recombinant I-type humanized collagen C1L4T and preparation method and application thereof
CN116554309A (en) Recombinant human III type collagen and preparation method and application thereof
CN113683678B (en) Recombinant I-type humanized collagen C1L2T and preparation method and application thereof
CN110627889B (en) Recombinant spider silk protein, preparation method and industrial application thereof
CN114805544B (en) Insulin lispro precursor, recombinant genetic engineering bacterium thereof and construction method thereof
CN117986355B (en) Recombinant humanized III type collagen and preparation method and application thereof
CN112266421B (en) Recombinant fusion protein and vaccine prepared from same and used for preventing PCV2 virus infection
WO2024119724A1 (en) Collagen peptide, preparation method therefor and use thereof
CN107312789A (en) A kind of preparation method of recombinant human bone morphogenesis protein
CN113735979A (en) Recombinant human platelet-derived factor (rhPDGF-BB) -Fc fusion protein and use thereof
JP6048959B2 (en) Production method of lysozyme
JP6206873B2 (en) Method for producing porcine lysozyme using secretory signal peptide derived from chicken lysozyme

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