CN115521373A - 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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- CN115521373A CN115521373A CN202210630810.XA CN202210630810A CN115521373A CN 115521373 A CN115521373 A CN 115521373A CN 202210630810 A CN202210630810 A CN 202210630810A CN 115521373 A CN115521373 A CN 115521373A
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- 102000012422 Collagen Type I Human genes 0.000 title claims abstract description 88
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
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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 characteristic triple-helix structure of collagen and a (Gly-X-Y) n repeated amino acid sequence and can be self-assembled to form collagen fibers with good appearance; 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 remarkably promote the proliferation, adhesion and migration of human skin fibroblasts; the recombinant humanized type I collagen is prepared by the expression of the genetic engineering bacteria, the process is simple and convenient, and the industrial production is easy to realize; the prepared triple-helix recombinant humanized type I collagen with high biological activity can be widely applied to the fields of skin repair dressing, implant, artificial skin, artificial bone, artificial cartilage, artificial eye cornea, biological materials, medical instruments, cosmetics, health 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, and accounts for about 30% of the total protein. The collagen has a characteristic triple-helix structure and can be self-assembled to form collagen fibers with good appearance. The collagen fibers are further assembled to form a fiber lattice structure, which provides mechanical strength to the connective tissue and a suitable environment for cell growth. To date, at least 40 more genes encoding collagen chains have been discovered. 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 a main component of connective tissues such as skin, bone, tendon, and the like.
The biological material with type I collagen as main material is widely used in tissue engineering, regenerative medicine and other fields. Type I collagen is currently extracted mainly by animal tissue. Different treatment methods and processes such as acid, alkali or protease have been developed to extract type I collagen from different tissues such as skin, tendon and bone of animals such as cattle and pig. However, the animal extraction method is easy to destroy the triple helix structure and the biological activity of the collagen, and has the risks of virus transmission and the like. The recombinant collagen prepared by the genetic engineering technology can overcome the risks of batch quality difference, virus infection hidden danger and the like of animal-derived collagen, and thus becomes a research hotspot gradually. Transgenic plants, mammalian cells, bacteria, yeast 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 cycle, and are difficult to meet the industrial requirements. 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.
For example, chinese patent CN110903383A discloses a recombinant human type I collagen, a coding gene, an engineering bacterium and its application in preparing a collagen preparation, wherein the amino acid sequence is peptide GSKGDTGEPGPVGVQGPPGPAGEEGKRGARGEP repeated 9 times; chinese patent CN113637068A provides a recombinant I-type humanized collagen C1L5T, a preparation method and application thereof, and the amino acid sequence of the recombinant I-type humanized collagen C1L5T comprises a peptide section GEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPTGPPGPRGRTGDAGPVGPPGPPGPPGPPGPP and a terminal sequence peptide section GAPGPCCGG of human type I collagen. Chinese patent CN105061589A provides a recombinant human type I collagen, which comprises 660-964 peptide segment of human type I collagen alpha 1 chain, 15 amino acids at 680-694 position at N end, 15 amino acids at 950-964 position at C end and 3 groups of GER amino acid triplets. However, the recombinant type I collagen prepared by the methods lacks the characteristic triple-helical structure, fibrous morphology and biological function of the collagen.
Aiming at the technical problems, the invention provides triple-helix recombinant humanized type I collagen, a preparation method and application thereof. The recombinant humanized type I collagen has a characteristic triple-helical structure of collagen, strictly complies with Gly-X-Y repetitive sequence by 100%, and can be self-assembled to form collagen fibers with good appearance; the recombinant humanized type I collagen has important functional sites of the human type I collagen, shows excellent biocompatibility and bioactivity, and can remarkably promote the proliferation, adhesion and migration of human skin fibroblasts; the recombinant humanized type I collagen is prepared by gene engineering bacteria expression and precursor collagen enzymolysis, the process is simple, the operation is convenient, and the industrial production is easy to realize; the recombinant humanized type I collagen can be widely applied to the fields of skin repair dressings, implants, artificial skin, artificial bones, artificial cartilages, artificial eye corneas, biological materials, medical instruments, cosmetics, health products, foods and the like.
Disclosure of Invention
In order to solve the technical problems, the invention firstly aims to provide the triple-helix recombinant humanized type I collagen which is collagen THRCI-1 or collagen THRCI-2, wherein 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 by treating precursor collagen with protease; the amino acid sequence of the precursor collagen 1 corresponding to the collagen THRCI-1 is shown as SEQ ID NO.3, and the amino acid sequence of the precursor collagen 2 corresponding to the 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 as SEQ ID No.5, and the gene sequence of the precursor collagen 2 corresponding to the collagen THRCI-2 is shown as SEQ ID No. 6.
Preferably, the protease comprises pepsin, trypsin, papain, bromelain, and thrombin.
The invention also provides a recombinant vector or recombinant gene engineering bacterium carrying the gene.
Preferably, the vector comprises pCold or pET.
Preferably, the engineering bacteria is 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 gene engineering bacteria constructed in the step (2), collecting bacterial precipitation, crushing to obtain supernatant, and purifying to obtain precursor collagen;
(4) And (4) 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) includes pepsin, trypsin, papain, bromelain, and thrombin.
The invention also provides application of the triple-helix recombinant humanized type I collagen in preparing skin repair dressings, implants, artificial skin, artificial bones, artificial cartilages, artificial eye corneas, biological materials, medical instruments, cosmetics, health products and foods.
Compared with the prior art, the invention has the beneficial effects that: (1) the invention provides triple-helix recombinant humanized type I collagen, and the amino acid sequence of the collagen is derived from human type I collagen alpha 1 Compared with the type I collagen from animal sources, the chain has the advantages of no virus transmission hidden danger, low immunogenicity and the like; (2) the recombinant humanized type I collagen provided by the invention has a characteristic triple-helical structure 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 which have good appearance and are 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, good biocompatibility and high bioactivity, and can remarkably promote the proliferation, adhesion and migration of human skin fibroblasts (HFF-1); (5) the genetic engineering bacteria expression system of the triple-helix recombinant humanized type I collagen provided by the invention has simple and convenient preparation process and is easy to realize large-scale production; (6) the triple-helix recombinant humanized type I collagen with high biological activity prepared by the invention can be widely applied to skin repair dressings, implants, artificial skin, artificial bones and artificial soft tissuesBone, artificial cornea, biological material, medical appliance, cosmetics, health-care product and food.
Drawings
The above and/or additional aspects and advantages of the present invention will become 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 chart of triple-helix recombinant humanized type I collagen
FIG. 2 is the determination of the secondary structure of triple helix recombinant humanized type I collagen
a, b and c are respectively a circular dichroism chart, a circular dichroism thermal change curve and a first derivative (d theta/dT) of the thermal change curve of the THRCI-1; d, e and f are respectively a circular dichroism chart, a circular dichroism thermal change curve and a first derivative (d theta/dT) of the thermal change curve of the THRCI-2
FIG. 3 is a Scanning Electron Microscope (SEM) image of triple helix recombinant humanized type I collagen
FIG. 4 is a diagram showing the cell proliferation of triple helix recombinant humanized type I collagen
FIG. 5 is a drawing showing the cell adhesion of triple helix recombinant humanized type I collagen
FIG. 6 is a graph of cell migration of triple helix recombinant humanized type I collagen
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.
The test 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 triple-helix recombinant humanized type I collagen THRCI-1 and the collagen THRCI-2 with high biological activity are respectively shown in SEQ ID NO.1 and SEQ ID NO.2, and the proteins are obtained by processing precursor collagen through 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. Determining the amino acid sequence of the precursor collagen as shown in SEQ ID NO.3 and SEQ ID NO. 4;
2. construction of expression strains of precursor collagen:
the gene sequences of the synthetic coded precursor collagen are shown as SEQ ID NO.5 and SEQ ID NO.6, plasmids introduced with the nucleic acid are constructed, and the successful synthesis of the plasmids is confirmed through DNA sequencing; transforming the plasmid into escherichia coli BL21-DE3 strain to obtain precursor collagen expression strain, adding glycerol into the successfully transformed strain, and storing in a refrigerator at-80 ℃;
3. preparing and purifying collagen:
adding 20 mu L of bacterial liquid into 200mL of LB liquid culture medium containing antibiotics, carrying out enrichment culture on the bacterial liquid by a constant-temperature shaking table at 37 ℃ overnight, transferring the bacterial liquid into 1L of LB liquid culture medium containing antibiotics according to the inoculum size of 2%, and continuing the amplification culture in the constant-temperature shaking table at 37 ℃; to be OD 600 Adjusting the temperature of a shaker to 25 ℃ when the value reaches the range of 0.8-1.0, adding 1mM IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration for induction expression, and culturing overnight at constant temperature; centrifuging the bacterial liquid in a low-temperature centrifuge at 3200rpm,4 ℃ for 30min, and collecting the thallus.
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., 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 precursor collagen, purifying to remove enzyme digestion products, and freeze-drying to obtain the triple-helix recombinant humanized type I collagen with high biological activity.
The highly bioactive triple-helical recombinant humanized type I collagen prepared in example 1 was identified by SDS-PAGE.
FIG. 1 shows that the prepared recombinant humanized type I collagen THRCI-1 and recombinant humanized type I collagen THRCI-2 are single strips, which indicates that the high-purity recombinant humanized type I collagen is successfully prepared. Due to the specific amino acid sequence and structure of collagen, the migration rate on SDS-PAGE is less than that of globular proteins of the same molecular weight.
Example 2 determination of Secondary Structure of recombinant humanized type I collagen
Circular dichroism is a common method for characterizing protein structure. 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) scanning was performed at 4 ℃ in a 1mm cuvette, with 1nm wavelength interval, and the residence time at each wavelength was 5 seconds. The thermal change curve was measured at 220nm and the temperature was from 4 ℃ to 70 ℃. The temperature increase rate was as follows: 4-20 deg.c (1 deg.c/min); 20-50 deg.C (0.3 deg.C/min); 50-70 deg.c (1 deg.c/min).
As shown in figure 2, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 both show characteristic absorption peaks of collagen near 225nm, which indicates that the recombinant humanized type I collagen forms a triple helix structure. The study of thermal change curves further shows that the recombinant humanized I-type collagen THRCI-1 and the recombinant humanized I-type collagen THRCI-2 form a stable triple-helix structure, and the thermal change temperatures are 40 ℃ and 42 ℃ respectively.
Example 3 recombinant humanized type I collagen assembly morphology
The 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, dropped in small amounts on a silicon wafer until completely dried, and the dried samples were subjected to a 2-minute gold spraying treatment before characterization with Hitachi S-4800 Scanning Electron Microscope (SEM).
As shown in figure 3, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 are self-assembled to form collagen fibers with good appearance and similarity to natural collagen.
Example 4 biological Activity of recombinant humanized type I collagen
(1) Cell proliferation assay
Add 100. Mu.L to 96-well plates at a cell density of 1X 10 5 Human skin fibroblasts per mL HFF-1, 37 ℃,5% CO 2 After 24h and 24h incubation, the culture medium in the 96-well plate was aspirated, the recombinant humanized type I collagen prepared in example 1 and the tissue-extracted bovine type I collagen were added to the 96-well plate in a collagen solution of 0.1mg/mL in a DMEM high-sugar medium, and the control group was added with only DMEM high-sugar medium and continued at 37 ℃ and 5% CO 2 Incubate in incubator for 1, 3, 5 days. The designed recombinant humanized I collagen is used for detecting the effect of promoting the human skin fibroblast HFF-1 proliferation by using cck-8.
As shown in figure 4, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 both have obvious effect of promoting the proliferation of human skin fibroblasts HFF-1 and are superior to bovine type I collagen extracted by tissues.
(2) Cell adhesion experiment
The recombinant humanized type I collagen prepared in example 1 was prepared into a collagen solution of 0.1mg/mL with PBS, heat denatured 1% BSA and a bovine type I collagen standard of 0.1mg/mL were used as a negative control and a positive control, respectively, and each of the above protein solutions was added to a 24-well plate and adsorbed at 4 ℃ for 24 hours, followed by aspiration of the solution. Human skin fibroblast HFF-1 cells were diluted to a density of 1X 10 with DMEM high-glucose medium 5 one/mL, 500. Mu.L of cells were added to each well, and cell adhesion was observed under a microscope after 6 hours.
As shown in figure 5, compared with BSA, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 both show good cell adhesion effect and have excellent cell adhesion performance.
(3) Cell migration assay
The cell migration promoting effect of the recombinant humanized type I collagen prepared in example 1 was measured by the cell scratch method. And (3) transversely drawing 3 lines in each hole at the back of the 6-hole plate by using a marker pen, wherein the line spacing is 0.5-1cm. 6 well plates were seeded with 2mL of cells at a density of 2.5X 10 5 one/mL HFF-1 cell to ensure the cell culture to reach 95-100% confluence rate after 24 h. After 24h, the orifice plate was aligned with a 10 μ L tip against a ruler, scratched by nudging it down longitudinally, and prepared as in example 1The recombinant humanized type I collagen is prepared into a collagen solution of 0.1mg/mL by a DMEM high-sugar culture medium, and the DMEM high-sugar culture medium and the bovine type I collagen of 0.1mg/mL are respectively used as a negative control and a positive control. The change in scratch area was recorded for 0h and 24h under an inverted fluorescence microscope.
As shown in FIG. 6, the recombinant humanized type I collagen THRCI-1 and the recombinant humanized type I collagen THRCI-2 both showed good cell migration promoting effect compared to the blank control group.
The experimental results show that the recombinant humanized type I collagen prepared by the invention has a characteristic triple helical structure of collagen and a (Gly-X-Y) n repeated amino acid sequence, and can be self-assembled to form collagen fibers with good appearance; the recombinant humanized type I collagen prepared by the invention has important functional sites of the human type I collagen, shows good biocompatibility and bioactivity, and can obviously promote the proliferation, adhesion and migration of human skin fibroblasts; the recombinant humanized type I collagen is prepared by the expression of the genetic engineering bacteria, the process is simple and convenient, and the industrial production is easy to realize; the triple-helix recombinant humanized type I collagen with high biological activity prepared by the invention can be widely applied to the fields of skin repair dressings, implants, artificial skin, artificial bones, artificial cartilages, artificial eye corneas, biological materials, medical instruments, cosmetics, health products, foods and the like.
The above description is only for details of a specific exemplary embodiment of the present invention, and it is obvious to those skilled in the art that various modifications and changes may be made in the present invention in the practical application process according to specific preparation conditions, and the present invention is not limited thereto. All that comes within the spirit and principle of the invention is to be understood as being within the scope of the invention.
Sequence listing
<110> collagen (Wuhan) Biotechnology Ltd
<120> triple-helix recombinant humanized type I collagen, preparation method and application thereof
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Claims (10)
1. The triple-helix recombinant humanized type I collagen is characterized by being collagen THRCI-1 or collagen THRCI-2, wherein the amino acid sequence of the collagen THRCI-1 is shown in SEQ ID No.1, and the amino acid sequence of the collagen THRCI-2 is shown in SEQ ID No. 2.
2. The triple-helical recombinant humanized type I collagen of claim 1, which is obtained by treating a precursor collagen with a protease; the amino acid sequence of the precursor collagen corresponding to the collagen THRCI-1 is shown as SEQ ID NO.3, and the amino acid sequence of the precursor collagen corresponding to the collagen THRCI-2 is shown as SEQ ID NO. 4.
3. The triple-helix recombinant humanized type I collagen according to claim 2, wherein the gene sequence of precursor collagen 1 corresponding to collagen THRCI-1 is shown in SEQ ID No.5, and the gene sequence of precursor collagen 2 corresponding to collagen THRCI-2 is shown in SEQ ID No. 6.
4. The triple-helix recombinant humanized type I collagen according to claim 2, characterized in that said proteases comprise pepsin, trypsin, papain, bromelain, thrombin.
5. A recombinant vector or a recombinant genetically engineered bacterium carrying the gene of claim 4.
6. The recombinant vector or the recombinant genetically engineered bacterium of claim 5, wherein the vector comprises pCold or pET.
7. The recombinant vector or recombinant genetically engineered bacterium of claim 5, wherein the engineered bacterium is Escherichia coli.
8. The method for preparing triple-helix recombinant humanized type I collagen according to claim 1, which comprises the following steps:
(1) Synthesizing a gene sequence encoding the precursor collagen of claim 3;
(2) Connecting the gene sequence in the step (1) with a vector, transforming bacteria, and constructing recombinant genetic engineering bacteria;
(3) Expressing the recombinant gene engineering bacteria constructed in the step (2), collecting bacterial precipitation, crushing to obtain supernatant, and purifying to obtain precursor collagen;
(4) And (4) adding protease to treat the precursor collagen obtained in the step (3), and purifying to obtain the triple-helix recombinant humanized type I collagen.
9. The method according to claim 8, wherein the protease in step (4) comprises pepsin, trypsin, papain, bromelain, and thrombin.
10. The use of the triple-helix recombinant humanized type I collagen of claim 1 in the preparation of skin repair dressings, implants, artificial skin, artificial bone, artificial cartilage, artificial eye cornea, biomaterials, medical devices, cosmetics, health products, and foods.
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