CN115286709A - Self-assembled triple-helix recombinant collagen and gel formed by crosslinking same - Google Patents

Self-assembled triple-helix recombinant collagen and gel formed by crosslinking same Download PDF

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CN115286709A
CN115286709A CN202210737029.2A CN202210737029A CN115286709A CN 115286709 A CN115286709 A CN 115286709A CN 202210737029 A CN202210737029 A CN 202210737029A CN 115286709 A CN115286709 A CN 115286709A
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collagen
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肖建喜
何会霞
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Lanzhou Biological Technology Development Co ltd
Lanzhou University
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Lanzhou Biological Technology Development Co ltd
Lanzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a self-assembled triple-helix recombinant collagen and a gel formed by crosslinking the same. The invention provides a novel compound having (Gly-Tyr-Tyr) m (Gly‑Xaa‑Yaa) n (Gly‑Tyr‑Tyr) m The amino acid sequence pattern of the collagen protein and the characteristic triple helix structure of the collagen protein, and can be self-assembled to form the nano-fiber with good appearance. The self-assembled triple-helix recombinant collagen can be covalently crosslinked to form hydrogel with good appearance and mechanical property, and can remarkably promote the proliferation and adhesion of human fibroblasts. The self-assembled triple-helix recombinant collagen has high biocompatibility and bioactivity, and can be widely applied to the fields of skin repair materials, osteochondral repair materials, tissue engineering, medical cosmetology, skin care products and the like.

Description

Self-assembled triple-helix recombinant collagen and gel formed by crosslinking same
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a self-assembled triple-helix recombinant collagen and a gel formed by crosslinking the same.
Background
Collagen is the main structural protein in animal extracellular matrix, is composed of repeated amino acid sequence of (Gly-Xaa-Yaa) n, and has a unique triple helix structure. The triple-helical structure is a core characteristic of collagen, and the triple-helical collagen is further assembled to form collagen fibers. The collagen fiber is the main component of different tissues and organs such as bones, skins and the like, can provide a biological scaffold for a human body, and participates in various life activities such as regulation of interaction of biomacromolecules, cell proliferation and adhesion, tissue regeneration, wound repair and the like. As an excellent biological material, the collagen has excellent properties such as high biocompatibility, low immunogenicity, tissue degradability and the like.
The collagen material most commonly used at present is mainly extracted from tissues such as tendon, bone, skin and ligament of animals by acid method, alkali method, salt method and enzyme method. However, there are problems of protein denaturation, structural destruction, low protein yield, etc. during the extraction process, and there may be a risk of immune reaction and disease transmission. In addition, there may be batch-to-batch variability. Although the above problems can be solved by using eukaryotic expression collagen systems such as transgenic plants and animal cells, they have the problems of high culture cost, long period, low expression level, difficulty in large-scale production and the like.
Microbial expression systems such as E.coli and yeast are used for expression and production of recombinant collagen. Chinese patent CN110903383A prepares a recombinant type I collagen through the expression of an escherichia coli system, and the amino acid sequence of the recombinant type I collagen comprises 9 repeated peptide segments GSKGDTGEPGPVGQGPPGPAGEGKRGARGEP. Chinese patent CN103122027A discloses a method for preparing recombinant collagen by using Escherichia coli as expression vector, its amino acid sequence includes 8 or 16 times repeated III type collagen peptide segment and partial II type collagen peptide segment; chinese patent CN105061589A prepares recombinant human type I collagen through pichia pastoris engineering bacteria expression, mainly including 660-964 peptide segment of human type I collagen alpha 1 chain. However, none of the recombinant collagens prepared by the above methods can form a triple-helical structure characteristic to collagen, and can also not self-assemble to form collagen fibers, thereby limiting the application of the recombinant collagens in biomaterials.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a self-assemblable triple-helical recombinant collagen having (Gly-Tyr-Tyr) m (Gly-Xaa-Yaa) n (Gly-Tyr-Tyr) m Sequence characteristics, wherein m is more than or equal to 1 and less than or equal to 10, and n is more than or equal to 10 and less than or equal to 400; the recombinant collagen has a characteristic triple-helix structure of the collagen and can be self-assembled to form nano-fibers with good appearance; the self-assembled triple-helix recombinant collagen is obtained by enzyme treatment after the precursor protein is expressed by an escherichia coli expression system, and the operation is simple and convenient, and the large-scale production is easy; the self-assembled triple-helix recombinant collagen has high biocompatibility and bioactivity; the self-assembled triple-helix recombinant collagen can be crosslinked to form hydrogel with good appearance and mechanical properties, and can remarkably promote the proliferation and adhesion of human fibroblasts; the self-assembled triple-helix recombinant collagen has uniform molecular weight and stable quality, and can be widely applied to skin repair materials, osteochondral repair materials, tissue engineering, medical cosmetology and skin care productsAnd the like. The method specifically comprises the following steps:
in a first aspect, the present invention provides a triple-helix recombinant collagen capable of self-assembly, wherein the sequence of the recombinant collagen is: (Gly-Tyr-Tyr) m (Gly-Xaa-Yaa) n (Gly-Tyr-Tyr) m (ii) a Wherein m is more than or equal to 1 and less than or equal to 10, and n is more than or equal to 10 and less than or equal to 400.
Preferably, said m is 1 or 2.
Preferably, the amino acid sequence of the recombinant collagen is shown in any one of SEQ ID NO. 1-3.
In a second aspect, the present invention provides a method for preparing the recombinant collagen protein of the first aspect, the method comprising: treating precursor collagen with protease; the amino acid sequence of the precursor collagen is shown in SEQ ID NO. 4-6.
Preferably, the method comprises the steps of:
(1) Synthesizing a gene sequence for coding the precursor collagen, and constructing a recombinant plasmid containing the gene sequence;
(2) Transforming the recombinant plasmid in the step (1) into escherichia coli to construct recombinant genetic engineering bacteria;
(3) Culturing and inducing expression of the recombinant genetic engineering bacteria constructed in the step (2), centrifugally collecting bacteria, crushing the bacteria, collecting supernatant, and purifying to obtain precursor collagen;
(4) And (4) carrying out protease treatment on the precursor collagen obtained in the step (3), and purifying to obtain the triple-helix recombinant collagen capable of covalent self-assembly.
Preferably, the protease includes, but is not limited to, thrombin, pepsin, trypsin.
Preferably, the gene sequence encoding the precursor collagen is shown in SEQ ID NO. 7-9.
Preferably, the plasmid comprises a pCold or pET plasmid.
Preferably, the engineered bacterium is escherichia coli, including but not limited to e.coli BL21, e.coli BL21 (DE 3), e.coli DH5a, or e.coli TOP10.
In a third aspect, the present invention provides a recombinant plasmid, a recombinant vector or a recombinant genetically engineered bacterium carrying the collagen gene of the second aspect.
Preferably, the plasmids include, but are not limited to, pCold and pET series plasmids.
Preferably, the engineered bacterium is escherichia coli, including but not limited to e.coli BL21, e.coli BL21 (DE 3), e.coli DH5a, or e.coli TOP10.
In a fourth aspect, the present invention provides a gel formed by crosslinking the recombinant collagen of the first aspect; the triple-helix recombinant collagen forms hydrogel with good appearance and mechanical property through crosslinking.
Preferably, the crosslinking agent includes, but is not limited to, tris (bipyridyl) ruthenium (II) chloride, fe 2+ /H 2 O 2 。
In a fifth aspect, the present invention provides a biomimetic material prepared from the recombinant collagen of the first aspect.
In a sixth aspect, the present invention provides an application of the recombinant collagen of the first aspect or the biomimetic material of the fifth aspect in preparing skin repair materials, osteochondral repair materials, tissue engineering, medical cosmetology, and skin care products.
The beneficial effects of the invention are as follows:
(1) The recombinant collagen of the invention has (Gly-Xaa-Yaa) n The repeated amino acid sequence mode and the triple helix structure of the collagen protein can better simulate the structure of natural collagen protein;
(2) The recombinant collagen can be spontaneously assembled to form a fibrous structure with good appearance;
(3) The recombinant collagen has good biocompatibility and bioactivity, can well promote cell proliferation and adhesion, and has biological functions similar to those of natural collagen;
(4) The recombinant collagen is obtained by enzyme treatment after the expression of precursor collagen by an escherichia coli expression system, is simple and convenient to operate and is easy for large-scale production;
(5) The self-assembled triple-helix recombinant collagen can be covalently crosslinked to form hydrogel with good appearance and mechanical properties, and can remarkably promote the proliferation and adhesion of human fibroblasts;
(6) The self-assembled triple-helix recombinant collagen is an excellent biological material and has wide application prospects in the fields of skin repair materials, osteochondral repair materials, tissue engineering, medical cosmetology, skin care products and the like.
Drawings
FIG. 1 SDS-PAGE and circular dichroism chromatogram characterization of recombinant collagen, wherein A is SDS-PAGE pattern of different recombinant collagen samples, MW: molecular weight standard (kDa), 1; b is a circular dichroism chart; c is a thermal change curve; d is the first derivative of the temperature-dependent curve (D (MRE)/dT);
FIG. 2 dynamic light scattering measurement of the hydrated particle size of the self-assembled polymer of recombinant collagen at different times; wherein a is the change of the THRC-1 hydrated particle size with time; b is the change of THRC-2 hydrated particle size with time; c is the change of THRC-3 hydrated particle size with time;
FIG. 3 is a scanning electron micrograph of a recombinant collagen assembly; wherein a1 and a2 are THRC-1; b1 and b2 are THRC-2; c1 and c2 are THRC-3;
FIG. 4 2 [ RuII (bpy) 3 ]Cl 2 A cross-linked recombinant collagen gel; wherein a1-c1 is a recombinant collagen solution; a2-c2 is addition [ RuII (bpy) 3 ]Cl 2 The recombinant collagen solution is obtained; a3-c3 is recombinant collagen gel formed by photo-crosslinking; a1 and a2 are THRC-1; b1 and b2 are THRC-2; c1 and c2 are THRC-3;
FIG. 5Fe 2+ /H 2 O 2 A cross-linked recombinant collagen gel; wherein a1-c1 is a recombinant collagen solution; a2-c2 is recombinant collagen gel formed by photo-crosslinking; a1 and a2 are THRC-1; b1 and b2 are THRC-2; c1 and c2 are THRC-3;
FIG. 6 FESEM image of recombinant collagen sponge; wherein a1 and a2 are THRC-1; b1 and b2 are THRC-2; c1-c2 are THRC-3; the inset shows a photograph of a collagen sponge of 8 mm in diameter and its pore size distribution;
FIG. 7 rheological properties of a recombinant collagen hydrogel; wherein a-c is strain dependent (ω =10rad s) -1 ) Oscillatory shear rheology of collagen gel; d-f is the oscillatory shear rheology of the frequency dependent (strain 1%) collagen gel; a. d is THRC-1; b. e is THRC-2; c. f is THRC-3;
FIG. 8 shows the cell experiments of recombinant collagen gel; wherein a is the cytotoxicity of the cell; b is cell proliferation; c is the cell adhesion capacity on different materials; d-g is cell adhesion and spreading; actin staining is red, nuclear staining is blue;
FIG. 9 dynamic light scattering measurement of hydrated particle size of self-assembled polymer of control collagen at different times;
FIG. 10 2 RuII (bpy) 3 ]Cl 2 A cross-linked control collagen solution; wherein a1-b1 is a control collagen solution; a2-b2 is addition [ RuII (bpy) 3 ]Cl 2 The latter control collagen solution; a3-b3 are control collagen solutions after photocrosslinking.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the specification, and it is obvious that the described embodiments are only a part of the present invention, and not all of the invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
EXAMPLE 1 preparation of self-assemblable triple-helical recombinant collagen
1. Designing a triple-helix recombinant collagen sequence:
designing the amino acid sequences of the triple-helix recombinant collagens (THRC-1, THRC-2 and THRC-3) capable of self-assembling and precursor collagens thereof, which are respectively shown as SEQ ID NO.1-3 and SEQ ID NO. 4-6;
2. constructing recombinant gene engineering bacteria:
synthesizing a gene sequence for coding precursor collagen, which is shown as SEQ ID NO. 7-9; introducing the nucleic acid into a vector to construct a plasmid containing the nucleic acid, and confirming the successful synthesis of the plasmid through DNA sequencing; transforming the plasmid into a colon bacillus BL21-DE3 strain to obtain an expression strain of precursor collagen; adding the successfully converted strains into glycerol, and storing in a refrigerator at the temperature of-80 ℃;3. preparing triple-helix recombinant collagen:
culturing the expression strain at 37 ℃ for 8 hours, adding 1mM IPTG, cooling to 25 ℃, culturing for 10 hours, cooling to 20 ℃, continuously culturing for 8 hours, and centrifugally collecting thalli; dissolving thallus with buffer solution (20 mM sodium phosphate, 0.5M sodium chloride, pH 7.4), crushing cell, centrifuging the crushed suspension again, and collecting supernatant as crude protein solution; purifying the supernatant by using a nickel ion affinity column to obtain precursor collagen; adjusting pH of the solution to 2-3, adding pepsin, and standing for 16 hr; purifying the solution treated by pepsin to obtain triple-helix recombinant collagen, freeze-drying, and storing in a refrigerator at-20 ℃.
A small amount of the lyophilized powder was weighed, dissolved in water, and the purity of the protein was determined by SDS-PAGE. In FIG. 1, A is a graph showing that single bands were detected in the prepared recombinant collagens (THRC-1, THRC-2 and THRC-3) by SDS-PAGE, indicating that the high-purity recombinant collagen was successfully prepared.
Example 2 structural characterization of recombinant collagen
Recombinant collagens (THRC-1, THRC-2 and THRC-3) were prepared as in example 1, and prepared in a 1mg/mL solution with 10mM PBS buffer. The samples were equilibrated at 4 ℃ for at least 24 hours prior to testing. The thickness of the cuvette is 1mm, the wavelength scanning range is 210-260nm, and the interval of each step is 0.5nm. And (3) gradually raising the temperature within the temperature range of 4-60 ℃, and detecting the CD intensity at 220nm in real time. The sample was equilibrated at each temperature for 8s and the temperature was raised at a rate of 10 deg.C/h.
The positive absorption peak near 220nm of the CD spectrogram is a characteristic peak of the triple-helical structure of the collagen. All recombinant collagen samples (THRC-1, THRC-2 and THRC-3) had a positive peak at 220nm, indicating that they all formed the characteristic triple-helical structure of collagen (shown in B in FIG. 1). The thermal stability of the samples was examined by detecting the change in CD peak at 220nm with temperature, and the thermal change temperatures of the recombinant collagen THRC-1, THRC-2 and THRC-3 were 33.5 deg.C, 33.7 deg.C and 32.6 deg.C, respectively (shown in FIG. 1 as C-D). CD results indicate that recombinant collagens (THRC-1, THRC-2 and THRC-3) all form a stable triple helix structure.
Example 3 self-Assembly of triple helix recombinant collagen
1. Particle size distribution of triple-helix recombinant collagen solution
Triple-helical recombinant collagens THRC-1, THRC-2 and THRC-3 were prepared according to the method of example 1, and the hydrated particle size thereof was measured by dynamic light scattering, and the results are shown in FIG. 2. After 24 hours, the particle sizes of the triple-helix recombinant collagens are remarkably increased, and the triple-helix recombinant collagens are self-assembled.
2. Assembling morphology of triple-helix recombinant collagen
Triple-helical recombinant collagens THRC-1, THRC-2 and THRC-3 were prepared according to the method of example 1, the solution was dropped on a clean silicon wafer, left at room temperature for 24 hours, and the morphology of the triple-helical recombinant collagen assembly was characterized by Hitachi S-4800 scanning electron microscope (SEM, hitachi Limited, japan) (fig. 3). The triple-helix recombinant collagens THRC-1 (shown as a in figure 3), THRC-2 (shown as b in figure 3) and THRC-3 (shown as c in figure 3) can form good fibrous morphology.
Example 4 preparation of self-assemblable triple-helix recombinant collagen gel
1. Crosslinked gel under tris (bipyridine) ruthenium (II) chloride conditions
Triple-helical recombinant collagens THRC-1, THRC-2 and THRC-3 were prepared as in example 1, prepared as a 50mg/mL solution, and 3mM of tris (bipyridyl) ruthenium (II) chloride and 30mM of sodium persulfate salt (APS) were added. Curing with LED (430-480 nm, peak wavelength 455nm + -10nm, 1200mW cm) -2 ) Photocrosslinking was performed by irradiation for 20 seconds at a distance of 10-15mm above the protein solution. As shown in FIG. 4, the triple-helical recombinant collagen solution alone was colorless before the light irradiation (shown as a1 to c1 in FIG. 4), and the mixed solution after the addition of tris (bipyridyl) ruthenium (II) chloride and sodium persulfate salt was orange-colored (shown as a2 to c2 in FIG. 4); after illumination, the mixed solution turned into orange gelGlue (shown as a3-c3 in figure 4). The results show that the triple-helix recombinant collagens THRC-1, THRC-2 and THRC-3 are all photo-crosslinked into gel under the catalysis of tris (bipyridyl) ruthenium chloride (II) and the illumination condition.
2.Fe 2+ /H 2 O 2 Crosslinked gel under conditions
Triple-helical recombinant collagens THRC-1, THRC-2 and THRC-3 were prepared as in example 1, prepared into a 50mg/mL solution, and 2mM ferrous glucose sulfate and an equal volume of hydrogen peroxide (1%) were added and mixed well. As a result, as shown in FIG. 5, the triple-helical recombinant collagen solution alone was colorless (shown as a1-c1 in FIG. 5); the mixed solution of glucose ferrous sulfate and hydrogen peroxide is added to form a light yellow gel (a 2-c2 in figure 5). The results show that the triple-helix recombinant collagen proteins THRC-1, THRC-2 and THRC-3 are crosslinked to form gel under the catalysis of ferrous glucose sulfate and hydrogen peroxide.
Example 5 micro-morphology of triple-helical recombinant collagen gel
Gels of triple-helical recombinant collagen (THRC-1, THRC-2 and THRC-3) were prepared according to the method of example 4, pre-frozen overnight in a refrigerator at-20 ℃ and freeze-dried. And (3) characterizing the microstructure of the triple-helix recombinant collagen gel by using a Hitachi S-4800 scanning electron microscope. The aperture of each group was calculated from multiple FESEM images by pixel measurement software (E-muler) and the range and distribution of the aperture was described by randomly picking 50 data points per group, the results are shown in fig. 6. The triple-helix recombinant collagens (THRC-1, THRC-2 and THRC-3) can form a good net structure, and the average pore diameters are 105 +/-53.8 mu m,118 +/-32.6 mu m and 135 +/-69.3 mu m respectively.
Example 6 mechanical Properties of triple helix recombinant collagen gel
Gels of triple-helical recombinant collagens (THRC-1, THRC-2 and THRC-3) were prepared according to the method of example 4, and the mechanical properties of the gels were examined using an Anton Paar rheometer. The prepared recombinant collagen gel sample is placed in the middle of a sample stage with the diameter of 15 mm. The dynamic frequency sweep measurement was performed at a strain amplitude of 1%. We examined the rheological mechanical properties of the three recombinant collagen gels prepared and analyzed the storage modulus (elastic modulus G ') and loss modulus (viscous modulus G'). The results of strain-dependent oscillatory rheology showed that the THRC-1 gel, THRC-2 gel and THRC-3 gel were gel-thinned at strains of 96.1%,60.8% and 87.4%, respectively (shown as a-c in FIG. 7). The frequency-dependent (at 1% constant strain) oscillatory rheological results indicate that triple-helical recombinant collagen gels are dominated by storage modulus (G') (shown as d-f in fig. 7). At an angular frequency of 10rad/s, G' for THRC-1, THRC-2, and THRC-3 gels was 31.45Pa,38.59Pa, and 68.34Pa, respectively. These results indicate that the triple-helical recombinant collagen (THRC-1, THRC-2 and THRC-3) gels all have good mechanical properties.
Example 7 biocompatibility of triple-helical recombinant collagen gel
The CCK-8 method was used to evaluate the cytotoxicity of triple-helical recombinant collagen (THRC-1, THRC-2 and THRC-3) gels. Incubating HFF-1 cells in DMEM medium containing 15% bovine serum solution (5% 2 At 37 ℃ C. Samples of triple-helical recombinant collagen gels (0.01, 0.05,0.1 and 0.2 mg/ml) were prepared at different concentrations. mu.L HFF-1 cell suspension was added at 5X 10 per well 3 The density of individual cells was placed in 96-well cell culture plates and incubated for 24 hours (37 ℃,5% CO) 2 ) Thereafter, the cell culture solution was aspirated. 100 μ L of recombinant collagen gel solutions of different concentrations were added, and DMEM medium was added to the other wells as a control. After 24 hours of incubation to ensure cell adhesion, the plates were blotted clean and 100. Mu.L of 10% CCK-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt) was added to each well and incubated for 2 hours in a cell incubator. Absorbance at 450nm was measured using a Tecan Infinite F200/M200 multifunctional microplate reader (Tecan, mannedorf, switzerland).
The cytotoxicity test results are shown in a in fig. 8, the triple-helix recombinant collagen (THRC-1, THRC-2 and THRC-3) gels show good cell viability at different concentrations (0.01, 0.05,0.1 and 0.2 mg/ml), which indicates that the triple-helix recombinant collagen gels have excellent biocompatibility.
Example 8 biological Activity of triple helix recombinant collagen gel
1. Cell proliferation of triple-helical recombinant collagen gel
Proliferation of HFF-1 cells in triple helix recombinant collagen (THRC-1, THRC-2 and THRC-3) gel was detected by CCK-8 method. HFF-1 cells were plated at 5X 10 per well 3 Individual cells were seeded at density in 96-well plates. Cell proliferation was observed on days 1, 4, 7 and 11, during which time the medium was changed every two days, incubated until the date of the experiment, changed to basal medium containing 10% (v/v) CCK-8 reagent, and then continued for 6 hours. Absorbance of 100. Mu.L of the supernatant was measured at 450nm using a Tecan Infinite F200/M200 multifunctional microplate reader (Tecan, mannedorf, switzerland). As shown in the b in FIG. 8, the triple-helical recombinant collagen (THRC-1, THRC-2 and THRC-3) gels significantly promoted the proliferation of HFF-1 cells.
2. Cell adhesion of triple-helical recombinant collagen gel
Triple helix recombinant collagen (THRC-1, THRC-2, and THRC-3) gels were added to Nunclon Delta TC 96 microwell plates, with heat denatured BSA as a control. After washing the well plate 3 times with 10mM PBS buffer pH 7.4, 100. Mu.L of HFF-1 cell suspension (1X 10) was added 5 cell/mL), after 6-9 hours of incubation at 37 ℃, the unattached HFF-1 cells were washed with 10mM PBS buffer. Total deoxyribonucleic acid (DNA) quantification (Hoechst 33258) was used to compare the ability of materials to adhere to cells. In ultrapure water, adherent cells were lysed by repeated freeze-thawing. Then, hoechst33258 dye was added to the cell lysate to a final concentration of 5. Mu.g/mL, and the mixture was incubated in the dark for 1 hour. Finally, their fluorescence intensity at an emission wavelength of 465mm was measured at an excitation wavelength of 360mm using a microplate reader (Tecaminfamimite M200). Triplicate determinations were made.
As a result of the experiment, referring to the adhesion value of HFF-1 cells to native collagen as 100%, the adhesion ability of HFF-1 cells to BSA was 21.6% of that of native collagen, and the adhesion rates to THRC-1 gel, THRC-2 gel and THRC-3 gel were 76.5%,81.8% and 78.4%, respectively, as shown in FIG. 8 c. This result indicates that heat denatured BSA did not adhere well to HFF-1 cells, and in contrast, the triple-helical recombinant collagen (THRC-1, THRC-2, and THRC-3) gels showed good adhesion to HFF-1 cells.
3. Immunofluorescence reaction of triple-helix recombinant collagen gel
The collagen gel was washed with PBS until colorless, and the gel material was spread on tissue culture treated coverslips. The HFF-1 cell suspension was then incubated at 400cells/mm 2 Density was added to serum-free DMEM plates, incubated at 37 ℃ for 24 hours, washed three times with PBS, and non-adherent cells were removed. Then, the cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes, followed by blocking with 1% Bovine Serum Albumin (BSA) for 30 minutes. Cells were incubated with phalloidin-tetramethylrhodamine isothiocyanate at 37 ℃ for 1 hour, followed by addition of DAPI (Sigma-Aldrich) for 10 minutes at 37 ℃ for staining of the cellular actin cytoskeleton and nucleus. Images were taken at the fluorescence microscope.
The fluorescence microscopy results are shown in d-g in FIG. 8, and the cells of HFF-1 grown in Bovine Serum Albumin (BSA) are spherical, indicating that the cell adhesion ability is poor (shown in d in FIG. 8); in contrast, the fibroblasts grown in the gels THRC-1, THRC-2 and THRC-3 are mostly in a spread and adhered state (shown as e-g in FIG. 8). These results indicate that triple-helical recombinant collagen (THRC-1, THRC-2 and THRC-3) gels can significantly promote the adhesion and diffusion of HFF-1 cells. The results of cytotoxicity, proliferation and adhesion experiments show that the triple-helix recombinant collagen (THRC-1, THRC-2 and THRC-3) gel has excellent biocompatibility and bioactivity.
Comparative example 1 particle size distribution of control recombinant collagen solution
The recombinant collagen of the control group was prepared according to the method of example 1 (in comparison with the THRC-1 sequence, the recombinant collagen of control 1 did not contain tyrosine (Gly-Tyr-Tyr) at both ends; the recombinant collagen of control 2 did not have tyrosine residue (Gly-Tyr-Tyr) at the N-terminal and contained tyrosine (Gly-Lys-Tyr) at the C-terminal). The secondary structure was examined as in example 2. The hydrated particle size was measured by dynamic light scattering according to the particle size distribution of the reconstituted collagen solution in example 3. As a result, as shown in FIG. 9, the particle size of the recombinant collagen of control 1 was maintained at about 15nm and hardly changed after 24 hours. The particle size of the recombinant collagen of control 1 was always kept around 20 nm. These results indicate that recombinant collagen molecules without tyrosine present at both ends of the sequence have no driving force for assembly.
Control example 2 preparation of control recombinant collagen gel
The recombinant collagen of the control group was prepared according to the method of example 1 (in comparison with the THRC-1 sequence, the recombinant collagen of control 1 did not contain tyrosine (Gly-Tyr-Tyr) at both ends; the recombinant collagen of control 2 did not have tyrosine residue (Gly-Tyr-Tyr) at the N-terminal and contained tyrosine (Gly-Lys-Tyr) at the C-terminal). A control recombinant collagen hydrogel was prepared according to the method described in 1 in example 4. The results are shown in FIG. 10, in which a1-a3 are control 1 recombinant collagens, and b1-b3 are control 2 recombinant collagens; the results showed that the control recombinant collagen solution alone was colorless before the light irradiation (shown as a1-b1 in FIG. 10), and the mixed solution after the addition of tris (bipyridyl) ruthenium (II) chloride and sodium persulfate salt was orange-red (shown as a2-b2 in FIG. 10); after light irradiation, the solution remained an orange mixed solution (a 3-b3 in FIG. 10), and no gel was formed. The results show that the control recombinant collagen does not photocrosslink into gel under the catalysis of tris (bipyridyl) ruthenium (II) chloride and the illumination condition.
Sequence listing
<110> Lanzhou university
Lanzhou Biotechnology development Co., ltd
<120> a triple-helix recombinant collagen capable of self-assembly and gel formed by crosslinking the same
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Tyr Tyr Gly Leu Pro Gly Pro Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp
85 90 95
Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys Arg Gly Lys Gln Gly
100 105 110
Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp
115 120 125
Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Glu Arg Gly Glu Ala
130 135 140
Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly
145 150 155 160
Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro Gly Lys Asp Gly Lys
165 170 175
Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp
180 185 190
Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
195 200 205
Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly Lys Pro Gly Tyr Tyr
225 230 235 240
<210> 2
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly Tyr Tyr Gly Leu Pro Gly Pro Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp
85 90 95
Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys Arg Gly Lys Gln Gly
100 105 110
Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp
115 120 125
Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Glu Arg Gly Glu Ala
130 135 140
Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly
145 150 155 160
Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro Gly Lys Asp Gly Lys
165 170 175
Asp Gly Tyr Tyr Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp
180 185 190
Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
195 200 205
Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly Lys Pro Gly Tyr Tyr
225 230 235 240
<210> 3
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Tyr Tyr Gly Tyr Tyr Gly Pro Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp
85 90 95
Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys Arg Gly Lys Gln Gly
100 105 110
Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp
115 120 125
Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Glu Arg Gly Glu Ala
130 135 140
Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly
145 150 155 160
Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro Gly Lys Asp Gly Lys
165 170 175
Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp
180 185 190
Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
195 200 205
Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly Tyr Tyr Gly Tyr Tyr
225 230 235 240
<210> 4
<211> 325
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met His His His His His His Ala Asp Glu Gln Glu Glu Lys Ala Lys
1 5 10 15
Val Arg Thr Glu Leu Ile Gln Glu Leu Ala Gln Gly Leu Gly Gly Phe
20 25 30
Glu Lys Lys Asn Phe Pro Thr Leu Gly Asp Glu Asp Leu Asp His Thr
35 40 45
Tyr Met Thr Lys Leu Leu Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu
50 55 60
Asn Ser Trp Arg Lys Arg Leu Leu Lys Gly Ile Gln Asp His Ala Leu
65 70 75 80
Asp Leu Val Pro Arg Gly Tyr Tyr Gly Leu Pro Gly Pro Arg Gly Glu
85 90 95
Gln Gly Pro Thr Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln
100 105 110
Gly Leu Gln Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
115 120 125
Pro Ala Gly Pro Arg Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro
130 135 140
Thr Gly Leu Ala Gly Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr
145 150 155 160
Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly
165 170 175
Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys
180 185 190
Arg Gly Lys Gln Gly Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln
195 200 205
Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly
210 215 220
Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe
225 230 235 240
Pro Gly Glu Arg Gly Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro
245 250 255
Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly
260 265 270
Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys
275 280 285
Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp
290 295 300
Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly
305 310 315 320
Lys Pro Gly Tyr Tyr
325
<210> 5
<211> 325
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met His His His His His His Ala Asp Glu Gln Glu Glu Lys Ala Lys
1 5 10 15
Val Arg Thr Glu Leu Ile Gln Glu Leu Ala Gln Gly Leu Gly Gly Phe
20 25 30
Glu Lys Lys Asn Phe Pro Thr Leu Gly Asp Glu Asp Leu Asp His Thr
35 40 45
Tyr Met Thr Lys Leu Leu Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu
50 55 60
Asn Ser Trp Arg Lys Arg Leu Leu Lys Gly Ile Gln Asp His Ala Leu
65 70 75 80
Asp Leu Val Pro Arg Gly Tyr Tyr Gly Leu Pro Gly Pro Arg Gly Glu
85 90 95
Gln Gly Pro Thr Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln
100 105 110
Gly Leu Gln Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
115 120 125
Pro Ala Gly Pro Arg Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro
130 135 140
Thr Gly Leu Ala Gly Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr
145 150 155 160
Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly
165 170 175
Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys
180 185 190
Arg Gly Lys Gln Gly Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln
195 200 205
Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly
210 215 220
Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe
225 230 235 240
Pro Gly Glu Arg Gly Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro
245 250 255
Gly Lys Asp Gly Lys Asp Gly Tyr Tyr Gly Lys Asp Gly Leu Pro Gly
260 265 270
Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys
275 280 285
Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp
290 295 300
Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly
305 310 315 320
Lys Pro Gly Tyr Tyr
325
<210> 6
<211> 325
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met His His His His His His Ala Asp Glu Gln Glu Glu Lys Ala Lys
1 5 10 15
Val Arg Thr Glu Leu Ile Gln Glu Leu Ala Gln Gly Leu Gly Gly Phe
20 25 30
Glu Lys Lys Asn Phe Pro Thr Leu Gly Asp Glu Asp Leu Asp His Thr
35 40 45
Tyr Met Thr Lys Leu Leu Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu
50 55 60
Asn Ser Trp Arg Lys Arg Leu Leu Lys Gly Ile Gln Asp His Ala Leu
65 70 75 80
Asp Leu Val Pro Arg Gly Tyr Tyr Gly Tyr Tyr Gly Pro Arg Gly Glu
85 90 95
Gln Gly Pro Thr Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln
100 105 110
Gly Leu Gln Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
115 120 125
Pro Ala Gly Pro Arg Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro
130 135 140
Thr Gly Leu Ala Gly Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr
145 150 155 160
Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly
165 170 175
Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys
180 185 190
Arg Gly Lys Gln Gly Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln
195 200 205
Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly
210 215 220
Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe
225 230 235 240
Pro Gly Glu Arg Gly Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro
245 250 255
Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly
260 265 270
Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys
275 280 285
Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp
290 295 300
Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly
305 310 315 320
Tyr Tyr Gly Tyr Tyr
325
<210> 7
<211> 975
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgcatcacc accaccacca tgctgatgaa caagaggaaa aggcgaaagt gcgtaccgaa 60
ctgatccagg agctggcgca gggtctgggc ggctttgaaa aaaaaaactt cccgaccctg 120
ggtgatgaag atctggatca tacctatatg accaaactgc tgacctatct gcaagaacgt 180
gaacaggcag aaaacagctg gcgtaaacgt ctgctgaaag gtattcagga ccacgccctg 240
gatttggttc cgcgcggtta ctacggctta ccgggtccgc gcggtgagca ggggccgact 300
ggtccgaccg gtccggctgg tccgcgtggt ttacaaggtc tccaaggctt gcaaggtgag 360
cgtggtgaac aaggaccgac cggtccggcg ggtccgagag gtctgcaggg tgagcgcggg 420
gagcaaggcc caacgggtct ggcaggcaag gctggagagg cgggcgcgaa aggcgagact 480
ggtccggcag gtccgcaggg tccacgtggc gaacaaggcc cgcagggctt gccgggcaag 540
gacggcgagg cgggcgccca gggtcgtccg ggcaagcgcg gcaagcaggg ccagaaaggt 600
gagaagggtg agccgggcac ccagggtgcg aaaggcgatc gtggcgagac gggtcctgtt 660
ggtccgcgtg gtgaacgcgg cgaagcgggt ccagctggca aagatggcga gcgtggattc 720
ccgggtgagc gcggggtgga aggtcagaat ggtcaggatg gcctgccggg caaggacggc 780
aaagatggac aaaatggtaa agacggtttg ccgggcaagg acggcaagga cggccaaaac 840
ggcaaagacg gtttgccagg caaggatggc aaggacggtc aagatggtaa ggacggcctg 900
ccgggcaagg acggcaaaga tggtcttccg ggtaaggacg gtaaggacgg tcagccgggt 960
aaaccgggtt attac 975
<210> 8
<211> 975
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgcatcacc accaccacca tgctgatgaa caagaggaaa aagccaaggt gcgtaccgaa 60
ctgatccaag aactggccca gggccttggt ggtttcgaaa aaaaaaattt tccgaccctg 120
ggtgatgaag atttggatca cacctatatg accaaactgc tgacctatct gcaagagcgt 180
gaacaggcag aaaacagctg gcgtaaacgt ctgctgaaag gtattcagga ccatgcactg 240
gatttggttc cgcgtggtta ttacggcctg ccgggtccgc gcggcgaaca aggtccgacg 300
ggtccgaccg gtccagcagg cccgcgtggt ctgcaaggtc tgcagggttt gcaaggcgaa 360
agaggtgagc aaggaccgac cggtccagct ggtccgcgtg gtttacaagg tgagcgtggt 420
gagcagggtc cgacgggctt ggctggtaaa gcgggtgaag ccggtgcgaa gggtgagact 480
ggtccggcgg gtccacaggg cccgcgtggc gaacagggcc cacagggtct gccgggcaaa 540
gacggtgagg cgggtgcgca ggggcgtccg ggtaagcgcg gcaagcaggg tcaaaaaggc 600
gagaaaggtg agccgggcac gcagggcgcg aaaggtgatc gtggggaaac cggcccggtt 660
ggtccgcgcg gcgagcgcgg tgaggcgggt ccggctggca aggatggcga gcgcggcttc 720
ccgggtgagc gcggggtgga aggtcagaac ggccaggatg gcttgccggg taaggacggc 780
aaggacggct actacggcaa agatggactt ccgggtaagg atggcaagga cggccaaaac 840
ggtaaggacg gcttgccggg caaggacggc aaggacggcc aagatgggaa ggacgggtta 900
ccgggcaaag atggcaaaga cggcctgcct ggtaaggacg gcaaggacgg tcagccgggt 960
aaaccgggtt attac 975
<210> 9
<211> 975
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgcatcacc accaccacca tgctgatgaa caagaggaaa aagccaaagt gcgtaccgaa 60
ctgatccagg agctggctca gggtttaggt ggttttgaaa aaaaaaattt cccgaccctg 120
ggtgatgagg atctggacca cacctacatg accaaactgc tgacctatct gcaagaacgt 180
gaacaggcgg aaaacagctg gcgtaaacgt cttctgaaag gtattcagga ccatgcactg 240
gatctggttc cgcgtggtta ctatggttat tacggcccgc gtggtgaaca aggcccgacg 300
ggtccgaccg gcccggcggg cccgcgtggc ttgcagggct tgcaaggcct ccagggcgag 360
agaggcgagc agggtcccac tggcccggcg ggcccgcgcg gtttacaagg tgagagaggc 420
gagcagggtc caacgggtct ggcaggcaag gcaggcgaag cgggcgctaa gggtgagact 480
ggtccggctg gtccgcaggg cccgcgcggt gaacaaggtc cgcagggcct gcctggtaaa 540
gacggcgagg ccggtgcgca aggccgtccg ggcaagcgcg gtaagcaggg ccagaaaggt 600
gagaaaggtg agccgggtac ccagggagcc aagggcgacc gtggtgaaac cggtccggtt 660
ggtccgcgtg gtgagcgcgg tgaggcgggt ccagcgggca aagacggcga acgtgggttc 720
ccgggcgagc gcggggtgga aggtcaaaat ggccaggatg gtttgccggg taaggacggt 780
aaggacggtc aaaacggtaa ggatggcctg ccgggtaaag atggtaaaga cggccaaaac 840
ggtaaggacg gcttgccagg caaggatggc aaggacggcc aagatggcaa agacggcttg 900
ccgggtaagg atggcaagga cggcctaccg ggtaaggacg gtaaggatgg tcagccggga 960
tactacggtt attac 975

Claims (10)

1. A self-assemblable triple-helix recombinant collagen, said recombinant collagen having the sequence: (Gly-Tyr-Tyr) m (Gly-Xaa-Yaa) n (Gly-Tyr-Tyr) m (ii) a Wherein m is more than or equal to 1 and less than or equal to 10, and n is more than or equal to 10 and less than or equal to 400.
2. The recombinant collagen according to claim 1, wherein m is 1 or 2.
3. The recombinant collagen according to claim 2, wherein said recombinant collagen has an amino acid sequence as set forth in any one of SEQ ID No.1 to 3.
4. The method for producing recombinant collagen according to claim 3, wherein said method comprises: treating precursor collagen with protease; the amino acid sequence of the precursor collagen is shown in SEQ ID NO. 4-6.
5. The method of claim 4, wherein the method comprises the steps of:
(1) Synthesizing a gene sequence for coding the precursor collagen, and constructing a recombinant plasmid containing the gene sequence;
(2) Transforming the recombinant plasmid in the step (1) into escherichia coli to construct recombinant genetic engineering bacteria;
(3) Culturing and inducing expression of the recombinant genetic engineering bacteria constructed in the step (2), centrifugally collecting bacteria, crushing the bacteria, collecting supernatant, and purifying to obtain precursor collagen;
(4) Performing protease treatment on the precursor collagen obtained in the step (3), and purifying to obtain the triple-helix recombinant collagen capable of covalent self-assembly; the protease comprises thrombin, pepsin and trypsin.
6. The method of claim 5, wherein the gene encoding said precursor collagen has the sequence shown in SEQ ID No. 7-9.
7. A gel formed by crosslinking the recombinant collagen of any one of claims 1-3.
8. The gel of claim 7, wherein said cross-linking agent comprises tris (bipyridyl) ruthenium (II) chloride, fe 2+ /H 2 O 2 。
9. A biomimetic material prepared from the recombinant collagen of any one of claims 1-3.
10. Use of the recombinant collagen of any one of claims 1 to 3 or the biomimetic material of claim 9 for the preparation of skin repair materials, osteochondral repair materials, tissue engineering, medical cosmetology, skin care products.
CN202210737029.2A 2022-06-27 2022-06-27 Self-assembled triple-helix recombinant collagen and gel formed by crosslinking same Pending CN115286709A (en)

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