CN115521373B - Triple helix recombinant humanized type I collagen, preparation method and application thereof - Google Patents
Triple helix recombinant humanized type I collagen, preparation method and application thereof Download PDFInfo
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- CN115521373B CN115521373B CN202210630810.XA CN202210630810A CN115521373B CN 115521373 B CN115521373 B CN 115521373B CN 202210630810 A CN202210630810 A CN 202210630810A CN 115521373 B CN115521373 B CN 115521373B
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Abstract
The invention discloses a triple helix recombinant humanized type I collagen, a preparation method and application thereof, wherein the recombinant humanized type I collagen has a triple helix structure and a (Gly-X-Y) n repeated amino acid sequence which are characteristic of the collagen, and can be self-assembled to form collagen fibers with good morphology; the recombinant humanized type I collagen prepared by the invention has important functional sites of human type I collagen, shows good biocompatibility and bioactivity, and can obviously promote proliferation, adhesion and migration of human skin fibroblasts; the recombinant humanized type I collagen is prepared by expressing the genetically engineered bacteria, the process is simple and convenient, and the industrial production is easy to realize; the prepared three-helix recombinant humanized type I collagen with high bioactivity can be widely applied to the fields of skin repair dressing, implant, artificial skin, artificial bone, artificial cartilage, artificial cornea, biological material, medical instrument, cosmetics, health care products, food and the like.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to triple helix recombinant humanized type I collagen, a preparation method and application thereof.
Background
Collagen is the highest protein content in mammals, accounting for about 30% of the total protein. The collagen has a characteristic triple helix structure, and can self-assemble to form collagen fibers with good morphology. The collagen fibers are further assembled to form a fiber lattice structure, providing mechanical strength to connective tissue and providing a suitable environment for cell growth. Up to now, at least 40 more collagen chain-encoding genes have been found. These different collagen chains, combined in different ways, can form more than 20 different types of collagen molecules. Among them, type I collagen is the most abundant type of collagen in the human body, and is the main component of connective tissues such as skin, bone, tendon, etc.
The biological material with the type I collagen as the main raw material is widely applied in the fields of tissue engineering, regenerative medicine and the like. Type I collagen is currently mainly extracted by animal tissue. Different treatment methods and processes of acid, alkali or protease and the like have been developed to extract type I collagen from different tissues such as skin, tendon, bone and the like of animals such as cattle, pigs and the like. However, animal extraction methods tend to destroy the triple helix structure and bioactivity of collagen, with the risk of potential viral transmission hazards. The recombinant collagen prepared by the genetic engineering technology can overcome the risks of batch quality difference, virus infection hidden trouble and the like of animal-derived collagen, so that the recombinant collagen gradually becomes a research hot spot. Transgenic plants, mammalian cells, bacteria, yeasts and the like are utilized to produce recombinant collagen, but expression systems of transgenic plants, mammalian cells and the like have high production cost and long period, and are difficult to meet the industrialization requirement. In contrast, the escherichia coli and yeast expression system has the advantages of low cost, high expression level and the like, and is the preferred mode of industrial production.
Regarding collagen, many researches have been made in the art, for example, chinese patent CN110903383a discloses a recombinant human type I collagen, coding gene, engineering bacteria and its application in preparing collagen preparation, the amino acid sequence of which is 9 times repeated as peptide GSKGDTGEPGPVGVQGPPGPAGEEGKRGARGEP; chinese patent CN113637068a provides a recombinant type I humanized collagen C1L5T, a preparation method and use, and its amino acid sequence includes peptide GEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPTGPPGPRGRTGDAGPVGPPGPPGPPGPPGPP and terminal sequence peptide GAPGPCCGG of human type I collagen. Chinese patent CN105061589a provides a recombinant human type I collagen comprising a peptide stretch of human type I collagen α1 chain 660-964, 15 amino acids at position N-terminal 680-694, and 15 amino acids at position C-terminal 950-964 and a 3 group GER amino acid triplet. However, the recombinant type I collagen prepared by the methods lacks the three-helix structure, fiber morphology and biological function characteristic of collagen.
Aiming at the technical problems, the invention provides a triple helix recombinant humanized I-type collagen, a preparation method and application thereof. The recombinant humanized type I collagen has a triple helix structure which is characteristic of collagen, strictly adheres to Gly-X-Y repeated sequences by 100%, and can be self-assembled to form collagen fibers with good morphology; the recombinant humanized type I collagen has important functional sites of human type I collagen, shows excellent biocompatibility and bioactivity, and can remarkably promote proliferation, adhesion and migration of human skin fibroblasts; the recombinant humanized type I collagen is prepared by a genetic engineering bacterium expression and precursor collagen enzymolysis method, has a simple process, is convenient to operate, and is easy to realize industrial production; the recombinant humanized type I collagen can be widely applied to the fields of skin repair dressing, implant, artificial skin, artificial bone, artificial cartilage, artificial cornea, biological material, medical instrument, cosmetics, health care products, food and the like.
Disclosure of Invention
Aiming at the technical problems, the primary purpose of the invention is to provide triple helix recombinant humanized type I collagen, wherein the triple helix recombinant humanized type I collagen is collagen THRCI-1 or collagen THRCI-2, the amino acid sequence of the collagen THRCI-1 is shown as SEQ ID NO.1, and the amino acid sequence of the collagen THRCI-2 is shown as SEQ ID NO. 2.
Preferably, the recombinant humanized type I collagen is obtained after treating a precursor collagen with a protease; the amino acid sequence of precursor collagen 1 corresponding to collagen THRCI-1 is shown as SEQ ID NO.3, and the amino acid sequence of precursor collagen 2 corresponding to collagen THRCI-1 is shown as SEQ ID NO. 4.
Preferably, the gene sequence of the precursor collagen 1 corresponding to the collagen THRCI-1 is shown in SEQ ID NO.5, and the gene sequence of the precursor collagen 2 corresponding to the collagen THRCI-2 is shown in SEQ ID NO. 6.
Preferably, the protease comprises pepsin, trypsin, papain, bromelain, thrombin.
The invention also provides a recombinant vector or recombinant genetically engineered bacterium carrying the gene.
Preferably, the vector comprises pCold or pET.
Preferably, the engineering bacteria are escherichia coli.
The invention also provides a preparation method of the triple helix recombinant humanized type I collagen, which comprises the following steps:
(1) Synthesizing a gene sequence encoding the precursor collagen;
(2) Connecting the gene sequence in the step (1) with a vector, transforming bacteria, and constructing recombinant genetic engineering bacteria;
(3) Expressing the recombinant genetically engineered bacteria constructed in the step (2), collecting bacterial precipitate, crushing to obtain supernatant, and purifying to obtain precursor collagen;
(4) And (3) adding protease to treat the precursor collagen obtained in the step (3), and purifying to obtain the triple helix recombinant humanized type I collagen.
Preferably, the protease in step (4) comprises pepsin, trypsin, papain, bromelain, thrombin.
The invention also provides application of the triple helix recombinant humanized type I collagen in preparing skin repair dressing, implant, artificial skin, artificial bone, artificial cartilage, artificial cornea, biological material, medical instrument, cosmetics, health care product and food.
Compared with the prior art, the invention has the beneficial effects that: ① The invention provides a triple helix recombinant humanized type I collagen, wherein the amino acid sequence of the collagen is derived from a human type I collagen alpha 1 chain, and compared with the type I collagen from animal sources, the triple helix recombinant humanized type I collagen has the advantages of no hidden danger of virus transmission, low immunogenicity and the like; ② The recombinant humanized type I collagen provided by the invention has a triple helix structure characteristic of collagen and a (Gly-X-Y) n repeated amino acid sequence; ③ The triple helix recombinant humanized type I collagen provided by the invention can be self-assembled to form collagen fibers with good morphology and similar to natural collagen; ④ The triple helix recombinant humanized type I collagen provided by the invention has important functional sites of human type I collagen, has good biocompatibility and high biological activity, and can obviously promote proliferation, adhesion and migration of human skin fibroblasts (HFF-1); ⑤ The three-helix recombinant humanized I-type collagen gene engineering bacterium expression system provided by the invention has the advantages that the preparation process is simple and convenient, and the large-scale production is easy to realize; ⑥ The three-helix recombinant humanized type I collagen with high bioactivity prepared by the invention can be widely applied to the fields of skin repair dressing, implant, artificial skin, artificial bone, artificial cartilage, artificial cornea, biological material, medical equipment, cosmetics, health care products, food and the like.
Drawings
The foregoing and/or additional aspects and advantages of the present invention will be apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings.
FIG. 1 is a SDS-PAGE identification of triple helix recombinant humanized type I collagen
FIG. 2 is a two-level structure assay of triple helix recombinant humanized type I collagen
A, b, c are respectively a circular dichroism spectrum of THRCI-1, a circular dichroism spectrum thermal deformation curve and a first derivative (dθ/dT) of the thermal deformation curve; d, e, f are THRCI-2 circular dichroism spectrum, circular dichroism spectrum thermal curve and first derivative of thermal curve (dθ/dT), respectively
FIG. 3 is a Scanning Electron Microscope (SEM) image of triple helix recombinant humanized type I collagen
FIG. 4 is a diagram showing the proliferation of cells of triple helix recombinant humanized type I collagen
FIG. 5 is a diagram showing cell adhesion of triple helix recombinant humanized type I collagen
FIG. 6 is a cell migration diagram of triple helix recombinant humanized type I collagen
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand. The scope of the invention is not limited to the examples described below.
The experimental materials in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of recombinant humanized type I collagen
The amino acid sequences of the high-bioactivity triple helix recombinant humanized type I collagen THRCI-1 and the collagen THRCI-2 are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the protein is obtained by treating precursor collagen with protease; the amino acid sequence of the precursor collagen 1 is shown as SEQ ID NO.3, the gene sequence is shown as SEQ ID NO.5, the amino acid sequence of the precursor collagen 2 is shown as SEQ ID NO.4, and the gene sequence is shown as SEQ ID NO. 6.
1. The amino acid sequence of the precursor collagen is determined to be shown as SEQ ID NO.3 and SEQ ID NO. 4;
2. Construction of precursor collagen expression strains:
Synthesizing a gene sequence of the precursor collagen as shown in SEQ ID NO.5 and SEQ ID NO.6, constructing a plasmid for introducing the nucleic acid, and confirming successful synthesis of the plasmid through DNA sequencing; transforming the plasmid into an escherichia coli BL21-DE3 strain to obtain a precursor collagen expression strain, adding glycerol into the strain which is transformed successfully, and storing the strain in a refrigerator at-80 ℃;
3. preparation and purification of collagen:
Adding 20 mu L of bacterial liquid into 200mL of LB liquid medium containing antibiotics, carrying out enrichment culture on the bacterial liquid by a shaking table at a constant temperature of 37 ℃ overnight, transferring the bacterial liquid into 1L of LB liquid medium containing the antibiotics according to 2% of inoculation amount, and continuing the enrichment culture in the shaking table at the constant temperature of 37 ℃; when the OD 600 value reaches 0.8-1.0, adjusting the temperature of the shaking table to 25 ℃, adding 1mM IPTG with the final concentration for induction expression, and culturing at constant temperature overnight; centrifuging the bacterial liquid in a low-temperature centrifuge at 3200rpm and 4 ℃ for 30min, and collecting bacterial cells.
The cells were dissolved in a buffer (20 mM sodium phosphate buffer, 20mM imidazole, 0.5M sodium chloride, pH 7.4), disrupted by a high-pressure homogenizer, the disrupted suspension was centrifuged again, and the supernatant, i.e., a crude protein solution, was further purified by a nickel ion affinity column to obtain a precursor collagen. And (3) adding pepsin with the final concentration of 0.008mg/mL to treat the precursor collagen, purifying to remove enzyme digestion products, and freeze-drying to obtain the triple helix recombinant humanized type I collagen with high bioactivity.
The highly bioactive triple helix recombinant humanized type I collagen prepared in example 1 was subjected to SDS-PAGE identification.
FIG. 1 shows that the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 prepared are single bands, indicating that the recombinant humanized type I collagen with high purity is successfully prepared. Due to the specific amino acid sequence and structure of collagen, migration rate on SDS-PAGE is less than that of globular proteins of the same molecular weight.
EXAMPLE 2 determination of the secondary Structure of recombinant humanized type I collagen
Round dichroism is a common method used to characterize protein structures. The recombinant humanized type I collagen prepared in example 1 was prepared as a 1mg/mL solution, and circular dichromatic full wavelength (190-260 nm) scans were performed at 4℃using a 1mm cuvette, with 1nm wavelength intervals, and 5s at each wavelength. The thermal profile was measured at 220nm and the temperature was from 4℃to 70 ℃. The rate of temperature increase is as follows: 4 ℃ -20 ℃ (1 ℃/min); 20 ℃ -50 ℃ (0.3 ℃/min); 50 ℃ -70 ℃ (1 ℃/min).
As shown in FIG. 2, both the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 showed an absorption peak characteristic of collagen around 225nm, indicating that the recombinant humanized type I collagen forms a triple helix structure. Thermal profile studies further showed that the recombinant humanized type I collagen THRCI-1 and recombinant humanized type I collagen THRCI-2 formed stable triple helix structures with thermal temperatures of 40℃and 42℃respectively.
EXAMPLE 3 Assembly morphology of recombinant humanized type I collagen
Recombinant humanized type I collagen THRCI-1 and recombinant humanized type I collagen THRCI-2 prepared in example 1 were dissolved with 20mM PB to a final concentration of 3mg/mL, left at 4℃for at least 12hrs, a small amount of the droplets were taken and placed on a silicon wafer until completely dried, and the dried samples were subjected to a2 minute metal spraying treatment before characterization by Hitachi S-4800 Scanning Electron Microscope (SEM).
As shown in FIG. 3, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 self-assemble to form collagen fibers with good morphology and similar to natural collagen.
EXAMPLE 4 bioactivity of recombinant humanized type I collagen
(1) Cell proliferation assay
100 Mu L of human skin fibroblast HFF-1 with the cell density of 1X 10 5 per mL is added into a 96-well plate, the culture solution in the 96-well plate is sucked out after incubation for 24 hours at 37 ℃ and 5% CO 2, the recombinant humanized type I collagen prepared in the example 1 and the tissue extracted bovine type I collagen are prepared into a 0.1mg/mL collagen solution by using a DMEM high sugar culture medium, the collagen solution is added into the 96-well plate, a control group is only added with the DMEM high sugar culture medium, and the culture is continued to be incubated for 1, 3 and 5 days in a 5% CO 2 incubator at 37 ℃. The designed recombinant humanized I collagen is used for detecting the proliferation effect of human skin fibroblast HFF-1.
As shown in FIG. 4, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 have remarkable effect of promoting proliferation of human skin fibroblast HFF-1 and are superior to bovine type I collagen extracted from tissues.
(2) Cell adhesion experiments
The recombinant humanized type I collagen prepared in example 1 was prepared into a collagen solution of 0.1mg/mL using PBS, heat denatured 1% BSA and bovine type I collagen standard of 0.1mg/mL were used as a negative control and a positive control, respectively, each of the above protein solutions was added to a 24-well plate to be adsorbed at 4℃for 24 hours, and then the solution was aspirated. Human skin fibroblasts HFF-1 were diluted to a density of 1X 10 5 cells/mL with DMEM high-sugar medium, 500. Mu.L cells were added to each well, and cell adhesion was observed under a microscope after 6 hours.
As shown in fig. 5, both the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 showed good cell adhesion effect and excellent cell adhesion performance compared to BSA.
(3) Cell migration experiments
The cell migration promoting effect of the recombinant humanized type I collagen prepared in example 1 was measured by using a cell scratch method. 3 lines were drawn transversely per well behind a 6-well plate with a marker pen, the line spacing being 0.5-1cm. 2mL HFF-1 cells with a cell density of 2.5X10 5/mL were seeded in the 6-well plate to ensure that the cell culture was able to reach 95% -100% confluency after 24 h. After 24 hours, the well plate was aligned with a 10. Mu.L tip and a scratch was formed by gently pushing down in the longitudinal direction, and the recombinant humanized type I collagen prepared in example 1 was prepared with DMEM high-sugar medium to a collagen solution of 0.1mg/mL, DMEM high-sugar medium and 0.1mg/mL bovine type I collagen as a negative control and a positive control, respectively. The change in scratch area was recorded under an inverted fluorescence microscope for 0h and 24 h.
As shown in fig. 6, both the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 exhibited good cell migration promoting effect compared to the blank.
The experimental result shows that the recombinant humanized type I collagen prepared by the invention has a triple helix structure and (Gly-X-Y) n repeated amino acid sequence which are characteristic of collagen, and can be self-assembled to form collagen fibers with good morphology; the recombinant humanized type I collagen prepared by the invention has important functional sites of human type I collagen, shows good biocompatibility and bioactivity, and can obviously promote proliferation, adhesion and migration of human skin fibroblasts; the recombinant humanized type I collagen is prepared by expressing the genetically engineered bacteria, the process is simple and convenient, and the industrial production is easy to realize; the three-helix recombinant humanized type I collagen with high bioactivity prepared by the invention can be widely applied to the fields of skin repair dressing, implant, artificial skin, artificial bone, artificial cartilage, artificial cornea, biological material, medical equipment, cosmetics, health care products, food and the like.
The foregoing is merely illustrative of the details of the individual exemplary embodiments of the invention, and it will be apparent to those skilled in the art that the invention may be varied and modified in practice depending on the particular manufacturing conditions and is not intended to be limiting. It is intended that all such modifications within the spirit and principles of the invention be included within the scope of the invention.
Sequence listing
<110> Collagen (Wuhan) biotechnology Co.Ltd
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Claims (9)
1. The triple helix recombinant humanized type I collagen is collagen THRCI-1 or collagen THRCI-2, the amino acid sequence of the collagen THRCI-1 is shown as SEQ ID NO.1, and the amino acid sequence of the collagen THRCI-2 is shown as SEQ ID NO. 2.
2. A recombinant vector or recombinant genetically engineered bacterium carrying a gene sequence encoding the collagen amino acid sequence of claim 1.
3. The recombinant vector or recombinant genetically engineered bacterium of claim 2, wherein the vector comprises pCold or pET.
4. The recombinant vector or recombinant genetically engineered bacterium of claim 2, wherein the engineered bacterium is e.
5. The method for preparing triple helix recombinant humanized type I collagen according to claim 1, comprising the steps of:
(1) Synthesizing a gene sequence encoding a precursor collagen 1 corresponding to collagen THRCI-1 or a precursor collagen 2 corresponding to collagen THRCI-2 according to claim 1;
(2) Connecting the gene sequence in the step (1) with a vector, transforming bacteria, and constructing recombinant genetic engineering bacteria;
(3) Expressing the recombinant genetically engineered bacteria constructed in the step (2), collecting bacterial precipitate, crushing to obtain supernatant, and purifying to obtain precursor collagen;
(4) Adding protease to treat the precursor collagen obtained in the step (3), and purifying to obtain triple helix recombinant humanized type I collagen;
The gene sequence of precursor collagen 1 corresponding to collagen THRCI-1 in the step (1) is shown as SEQ ID NO.5, and the gene sequence of precursor collagen 2 corresponding to collagen THRCI-2 is shown as SEQ ID NO. 6; the amino acid sequence of precursor collagen 1 corresponding to collagen THRCI-1 is shown as SEQ ID NO.3, and the amino acid sequence of precursor collagen 2 corresponding to collagen THRCI-2 is shown as SEQ ID NO. 4;
The protease in the step (4) is pepsin.
6. Use of the triple helix recombinant humanized type I collagen according to claim 1 for the preparation of skin repair dressings, implants, biomaterials, medical devices.
7. The use of claim 6, wherein the biological material is artificial skin, artificial bone or artificial cornea.
8. The use of claim 7, wherein the artificial bone is artificial cartilage.
9. The use of the triple helix recombinant humanized type I collagen according to claim 1 in the preparation of cosmetics.
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| CN115991764B (en) * | 2023-03-21 | 2023-06-09 | 杭州因智拓生物技术有限公司 | Hydroxyproline modified recombinant III type humanized collagen and preparation method and application thereof |
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