A recombinant collagen type I and a preparation method and application thereof

Abstract

This invention belongs to the field of recombinant protein technology, specifically relating to a recombinant type I collagen, its preparation method, and its applications. The recombinant type I collagen is any one of the following: an amino acid sequence as shown in SEQ ID NO.1; an amino acid sequence obtained by amino acid substitution, insertion, replacement, addition, deletion, modification, or unit repetition based on the amino acid sequence shown in SEQ ID NO.1; or an amino acid sequence with greater than 80% identity to the amino acid sequence shown in SEQ ID NO.1. The recombinant type I collagen of this invention exhibits good biocompatibility, safety, and no significant cytotoxicity. It possesses extremely good proliferative, cell migration, and cell adhesion effects; it significantly promotes the proliferation of human umbilical vein endothelial cells and wound healing, and can be widely used in tissue engineering scaffolds, wound repair dressings, medical devices, and cosmetics, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein technology, specifically relating to a recombinant type I collagen, its preparation method, and its application. Background Technology

[0002] Collagen is the most abundant protein in mammalian tissues, accounting for 25% to 30% of the body's total protein content. It is also a key structural component of the extracellular matrix, widely present in all tissues and organs, providing strength, durability, and flexibility to tissues and organs such as skin, cartilage, tendons, ligaments, and internal organs. With its biodegradability, low immunogenicity, and ability to promote cell proliferation and differentiation, it is an ideal biomedical and cosmetic skincare material.

[0003] Traditional collagen mainly refers to collagen extracted from the skin and bones of pigs and cows. The source is relatively easy to obtain and the production cost is relatively low. However, the separation and purification process of collagen from this source is relatively complicated, and the separation of monomers is difficult. It may also carry viruses, posing certain safety risks.

[0004] Therefore, it is of great significance to provide a novel collagen with good biocompatibility and safety. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant type I collagen, which solves the problems existing in the prior art.

[0006] The technical solution adopted in this invention is: This invention provides a recombinant type I collagen, wherein the recombinant type I collagen is any one of the following: 1) The amino acid sequence as shown in SEQ ID NO.1; 2) An amino acid sequence obtained by replacing, inserting, substituting, adding, deleting, modifying, or repeating units based on the amino acid sequence shown in SEQ ID NO.1; 3) An amino acid sequence that has greater than 80% identity with the amino acid sequence shown in SEQ ID NO.1.

[0007] A second aspect of the present invention provides a nucleic acid molecule that encodes the recombinant type I collagen.

[0008] A third aspect of the present invention provides a recombinant expression vector comprising the aforementioned nucleic acid molecule.

[0009] Preferably, the starting vector of the recombinant expression vector is pPIC9K.

[0010] A fourth aspect of the present invention provides a recombinant strain comprising the recombinant expression vector described above.

[0011] Preferably, the recombinant strain originates from Pichia pastoris.

[0012] The fifth aspect of this invention provides a method for preparing the recombinant type I collagen, comprising the following steps: The recombinant strain expressing the recombinant type I collagen was inoculated into a fermentation medium for fermentation and culture. The bacterial cells were removed to obtain a supernatant containing the recombinant type I collagen. The supernatant was then separated and purified to obtain the recombinant type I collagen.

[0013] Preferably, the separation and purification method is at least one of salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

[0014] The sixth aspect of the present invention provides an application of the recombinant type I collagen, the nucleic acid molecule, the recombinant expression vector and / or the recombinant strain, wherein the application refers to the use in the preparation of cosmetics, health products, medical devices and / or tissue engineering materials.

[0015] Preferably, the medical device includes medical dressings, medical spray dressings, medical gel dressings, or medical freeze-dried powder dressings; The tissue engineering materials include subcutaneous fillers, artificial bone, artificial skin, oral absorbable biomembranes, bone implants, vascular stents, hemostatic agents, procoagulants, intercellular matrix scaffolds, or collagen sponges.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a recombinant type I collagen, which is any one of the following: 1) the amino acid sequence shown in SEQ ID NO.1; 2) an amino acid sequence obtained by amino acid substitution, insertion, replacement, addition, deletion, modification, or unit repetition based on the amino acid sequence shown in SEQ ID NO.1; 3) an amino acid sequence with greater than 80% identity to the amino acid sequence shown in SEQ ID NO.1. The recombinant type I collagen of this invention is derived from a truncated segment of the α1 chain of natural human type I collagen, and has undergone amino acid substitution. It exhibits good biocompatibility, safety, and no significant cytotoxicity. It possesses extremely good proliferative, cell migration, and cell adhesion effects; furthermore, it significantly promotes the proliferation of human umbilical vein endothelial cells and wound healing, and can be widely used in tissue engineering scaffolds, wound repair dressings, medical devices, and cosmetics, showing broad application prospects.

[0017] This invention also provides a method for preparing the recombinant type I collagen, namely: inserting a nucleotide sequence encoding the amino acid sequence into a vector to obtain a recombinant expression vector; then introducing the recombinant expression vector into host cells for culture; and finally obtaining recombinant type I collagen through an ion exchange purification process, achieving a high protein purity (above 99%), thus solving the problems of low expression levels, complex purification, and insufficient activity of recombinant type I collagen. At the same time, the yeast expression system used in this invention has a lower risk of immune rejection and avoids the potential disease transmission risks associated with traditional animal-derived collagen. Attached Figure Description

[0018] Figure 1 This is a map of the recombinant plasmid vector.

[0019] Figure 2 This is an SDS-PAGE image of the supernatant after fermentation of recombinant type I collagen.

[0020] Figure 3 SDS-PAGE images of recombinant type I collagen before and after purification. Before lane: Recombinant type I collagen without cation exchange column chromatography; After lane: Recombinant type I collagen after cation exchange column chromatography.

[0021] Figure 4 The effect of recombinant type I collagen on cell viability.

[0022] Figure 5 This is a cell migration diagram of recombinant type I collagen.

[0023] Figure 6 The attached diagram shows the cell adhesion of recombinant type I collagen. Detailed Implementation

[0024] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0025] The inventive concept of this invention is as follows: Recombinant collagen is produced by introducing collagen genes into cells through genetic engineering, and then using cellular expression and synthesis to produce collagen. Recombinant collagen has advantages such as high production controllability, regulated structure and function, avoidance of animal-derived infectious disease risks, reduced dependence on animal resources, high purity, and sustainable production, and is gradually becoming a research hotspot.

[0026] In recent years, the production of recombinant type I collagen using genetic engineering technology has become an important research direction. The preparation process typically involves: obtaining the gene encoding the α1 chain of human type I collagen through gene synthesis technology, cloning it into a suitable expression vector, and then transforming it into a prokaryotic or eukaryotic cell system for heterologous expression. Currently, commonly used expression systems include Escherichia coli, mammalian cells, insect cells, plant cells, and yeast cells. Among these, the yeast expression system has become the preferred solution for producing recombinant type I collagen due to its outstanding advantages such as high protein expression efficiency, convenient genetic modification, low production cost, and ability to achieve high-density fermentation.

[0027] However, in actual production and application, the production of recombinant type I collagen faces the following problems: how to design a reasonable gene sequence, select a suitable expression system, how to increase the expression level during fermentation, and the complexity of purification processes, which limit its practical application. Based on this, this invention provides a novel recombinant type I collagen and its preparation method. The research and development idea of ​​this invention is:

[0028] Recombinant type I collagen was formed by replacing amino acids in a truncated protein segment (positions 299-691) from the α1 chain of natural human type I collagen. A vector containing a nucleic acid molecule encoding recombinant type I collagen was introduced into host cells, and efficient secretion-induced expression was carried out in host cell (especially commercial Pichia pastoris) expression systems to finally obtain a recombinant type I collagen with high protein expression levels, which has good biological activities to promote cell proliferation, migration and adhesion.

[0029] Specifically, the preparation method of recombinant type I collagen includes: The nucleotide sequence encoding the amino acid sequence is inserted into the expression vector to obtain a recombinant plasmid vector. The recombinant plasmid vector is then introduced into the host cells for culture to induce the expression of recombinant type I collagen. The recombinant type I collagen is then separated and purified to obtain the recombinant type I collagen.

[0030] In this invention, the host cell can be a prokaryotic cell or a eukaryotic cell, including Escherichia coli, Bacillus subtilis or Bacillus licheniformis, Pichia pastoris, Saccharomyces cerevisiae, animal cells or plant cells; preferably Escherichia coli and Pichia pastoris; more preferably Pichia pastoris.

[0031] Recombinant type I collagen is expressed either constitutively or inducibly, or both. Induced expression uses isopropyl β-D-thiogalactoside (IPTG), β-galactoside, methanol, or ethanol as the inducing agent.

[0032] The separation and purification methods of this invention employ one or more of the following combinations: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation. Preferably, a combination of chromatographic chromatography and membrane separation, or a combination of ion exchange chromatography and membrane separation, is used.

[0033] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0034] Example 1 A recombinant type I collagen, the specific preparation method of which is as follows: Using whole-genome synthesis technology, the amino acid sequence of the human type I collagen α1 chain gene with amino acid substitutions from position 299 to 691 (the specific sequence is shown in SEQ ID NO.1) was amplified and synthesized, and after codon optimization, the nucleotide sequence shown in SEQ NO.2 was obtained.

[0035] SEQ ID NO.1: GQMGPRGFPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGERGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGERGPSGPQGPGGPPGPPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGFPGERGGPGSRGF PGERGVAGPKGFPGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGERGAAGEPGFPGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGFPGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGFPGERGFPGERGVQGPP.

[0036] SEQ ID NO.2:

[0037] In this embodiment, the preparation method of recombinant type I collagen includes the following steps: 1. Preparation of shuttle plasmid and Pichia pastoris expression strain.

[0038] The amino acid sequence shown in SEQ ID NO.1 was optimized according to the codons of the Pichia pastoris expression system to obtain the nucleotide sequence shown in SEQ ID NO.2. The obtained nucleotide sequence was then synthesized by Qingke Biotechnology Co., Ltd., and designated as I0393. The synthesized nucleotide sequence was ligated into the pPIC9K plasmid to obtain the recombinant plasmid pPIC9KI0393. The vector map is shown below. Figure 1 .

[0039] The pPIC9KI0393 plasmid was electroporated into Pichia pastoris GS115 competent cells. High copy number screening was performed using G418 resistance as a selection marker, and the optimal transformant was selected to obtain the Pichia pastoris expression strain.

[0040] 2. Induction of target protein expression.

[0041] High-copy transformants selected through resistance screening were picked, and single colonies were transferred to BMGY liquid medium (1% yeast extract, 10% PPB, 1.34% YNB, 2% peptone, and 1% glycerol) and cultured at 30°C and 220 rpm for 24 h until the OD reached 10. Then, a 1% inoculum was added to BMGY liquid medium (1% yeast extract, 10% PPB, 1.34% YNB, 2% peptone, and 1% methanol) to induce the expression of the target protein.

[0042] The composition of BMMY medium is similar to that of BMGY, but methanol is added as an inducer at an initial concentration of 1% (v / v) during the first 24 hours of culture to promote the expression of the target protein. The culture is then continued for 48 hours at 30°C and 220 rpm. After fermentation, the mixture is centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected for subsequent protein separation and purification.

[0043] 3. Purification of recombinant type I collagen I0393.

[0044] The collected supernatant was treated with a 10KD ultrafiltration membrane and concentrated to 1 / 10 of its original volume. The concentrated sample could be stored at 4℃ for further purification. Then, the eluent was subjected to cation exchange column chromatography, and the collected eluent was ultrafiltered, the conductivity was reduced, and then lyophilized to obtain recombinant type I collagen I0393.

[0045] SDSPAGE was used to analyze recombinant type I collagen before and after fermentation and purification. The SDSPAGE electrophoresis images are shown below. Figure 2 and Figure 3 As shown.

[0046] Figure 2 The figure shows that the recombinant collagen expressed by Pichia pastoris is approximately 55 kDa, indicating that the protein expressed after fermentation is the recombinant type I collagen to be expressed in this example. Further identification was performed using mass spectrometry. Figure 2 The protein at the shown location has an amino acid sequence coverage of 91.3% with the recombinant type I collagen in this embodiment.

[0047] Figure 3 This is a DSPAGE electrophoresis image of recombinant type I collagen before and after purification. This image confirms that the protein after cation exchange is still the desired target protein.

[0048] Therefore, the above electrophoresis diagram illustrates that the method of this embodiment can achieve the expression and preparation of recombinant type I collagen. In this embodiment, the protein content of recombinant type I collagen I0393 was tested. The test results showed that the protein content was 1.3 g / L, and the purity reached 99%. It can be seen that the recombinant type I collagen prepared in this embodiment has a high protein content, which further illustrates that recombinant type I collagen can achieve efficient secretory expression in host cells (especially commercial Pichia pastoris) expression systems.

[0049] Example 2 The application of a recombinant type I collagen was evaluated through the following experiments to assess the biological functional activity of the recombinant type I collagen I0393 from Example 1 above: Experiment 1: Cytotoxicity experiment.

[0050] Human umbilical vein endothelial cells that have grown to 80% of the bottom area of ​​the culture flask were harvested, digested with 0.25% trypsin containing EDTA, and cultured in complete medium to achieve a cell density of 8 × 10⁻⁶ cells / mL. 3Cell suspension of cells / mL was prepared. 100 μL of cell suspension was seeded into 96-well culture plates and cultured in an incubator at 37°C and 5% CO2 saturated humidity. After 24 h of cell culture, the complete culture medium was aspirated. Different concentrations of recombinant type I collagen solution diluted in DMEM / F12 medium were added to the experimental groups to make final concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, and 5 mg / mL, with 5 replicates for each concentration. The control group consisted of cells cultured in DMEM / F12 medium, and the blank group consisted of cell-free DMEM / F12 medium. The cells were cultured in an incubator at 37°C and 5% CO2 saturated humidity for another 24 h. After aspirating the culture medium from each group, add 110 μL of a mixture of DMEM / F12 medium and CCK8, and incubate in a cell culture incubator for 4 h. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm. The mixture contains 100 μL of DMEM / F12 medium and 10 μL of CCK8.

[0051] Based on the average absorbance of each group, the cell viability was calculated using the following formula: % .

[0052] in, A Cell viability (%) a : Absorbance values ​​of the experimental group; b : Absorbance value of the control group; c : Absorbance value of the blank group.

[0053] The results are as follows Figure 4 As shown, compared with the 100% survival rate of the control group, the recombinant type I collagen prepared in this invention has a promoting effect on cell proliferation. As the concentration increases, the promoting effect first increases and then decreases. The promoting effect is most significant at a concentration of 3 mg / mL. 3 mg / mL was selected as the working concentration for subsequent functional experiments.

[0054] Experiment 2: Cell migration experiment.

[0055] Human umbilical vein endothelial cells that have grown to 80% of the culture dish area were harvested, digested with 0.25% trypsin (containing EDTA), and cultured in complete medium to achieve a cell density of 4 × 10⁻⁶ cells / mL. 5Cells were cultured at a concentration of 10 cells / mL. 2 mL of the cell suspension was seeded into 6-well plates and incubated at 37°C with 5% CO2 and saturated humidity. After 24 h of culture, a 10 μL pipette tip was used to gently push downwards along the wells, creating a longitudinal scratch. The culture medium was then completely aspirated, and the cells were washed three times with PBS to remove any loose cells. In the experimental group, 3 mg / mL I0393 diluted in serum-free DMEM / F12 medium was added (three replicates). The control group received an equal volume of serum-free DMEM / F12 medium. Cells were incubated at 37°C with 5% CO2 and saturated humidity for 0 h, 6 h, and 12 h, and photographed under a 10x microscope. Results are shown below. Figure 5 As shown, the central scratch area was cell-free at 0 h. After culturing in recombinant type I collagen solution for 6 h, human umbilical vein endothelial cells migrated to the central scratch area. After culturing in recombinant type I collagen solution for 12 h, human umbilical vein endothelial cells migrated to the central scratch area, and the scratch completely disappeared. The results indicate that the recombinant type I collagen prepared in Example 1 has the ability to promote the migration of human umbilical vein endothelial cells.

[0056] Experiment 3: Cell adhesion experiment.

[0057] The following solutions were uniformly coated onto each of the 24-well plates: The experimental group (I0393) was added with I0393 diluted with PBS at a concentration of 3 mg / mL; The control group (BSA) was supplemented with BSA diluted with PBS at a concentration of 3 mg / mL. The control group (no solution was added) All of the above are equipped with 3 duplicate holes.

[0058] The coated 24-well plates were incubated overnight at 4°C. Subsequently, the plates were blocked at room temperature using 1% heat-denatured BSA. After blocking, the plates were washed twice with PBS buffer to obtain the 24-well plates coated with each group's solution. Human umbilical vein endothelial cells that had grown to 80% of the culture dish area were digested with 0.25% trypsin (containing EDTA) and then cultured in complete medium to achieve a cell density of 3 × 10⁻⁶ cells / well. 5 Cells were cultured at a concentration of 1 cell / mL. 1 mL of this cell suspension was seeded into 24-well culture plates and incubated at 37°C with 5% CO2 and saturated humidity for 12 hours. The culture medium was removed, and the cells were washed three times with PBS. Next, the cells were fixed with 4% paraformaldehyde solution for 15 minutes. After fixation, the paraformaldehyde solution was discarded, and 0.1% crystal violet solution was added for staining for 20 minutes. After staining, the crystal violet solution was removed, and the morphological characteristics of the cells were observed and recorded under a 10x microscope. The results are as follows: Figure 6As shown, compared with the Control group and the BSA group, the recombinant type I collagen I0393 treatment group had a significant effect on cell adhesion.

[0059] Based on the results of experiments 1 to 3 above, the recombinant type I collagen expressed in this invention has the ability to promote the proliferation, migration and adhesion of human umbilical vein endothelial cells, thereby achieving wound repair. In addition, the recombinant type I collagen also contains natural amino acid sequences with multiple functional sites, has a uniform molecular weight, good solubility, and good cell adhesion and proliferation properties, and can be applied in multiple fields.

[0060] The recombinant type I collagen expressed in this invention can be used in the preparation of skin care products, health products, medical devices, and tissue engineering materials. Specifically, skin care products include cosmetics or invasive beauty products; medical devices include medical dressings, medical spray dressings, medical gel dressings, or medical lyophilized powder dressings; and tissue engineering materials include subcutaneous fillers, artificial bone, artificial skin, oral absorbable biomembranes, bone implants, vascular scaffolds, hemostatic agents, procoagulants, intercellular matrix scaffolds, or collagen sponges.

[0061] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A recombinant type I collagen, characterized in that, The recombinant type I collagen is any one of the following: 1) The amino acid sequence as shown in SEQ ID NO.1; 2) An amino acid sequence obtained by replacing, inserting, substituting, adding, deleting, modifying, or repeating units based on the amino acid sequence shown in SEQ ID NO.1; 3) An amino acid sequence that has greater than 80% identity with the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant type I collagen as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that, The starting vector for the recombinant expression vector is pPIC9K.

5. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the recombinant expression vector of claim 3.

6. The recombinant strain according to claim 5, characterized in that, The recombinant strain originated from Pichia pastoris.

7. The method for preparing recombinant type I collagen as described in claim 1, characterized in that, The steps are as follows: The recombinant strain expressing the recombinant type I collagen was inoculated into a fermentation medium for fermentation and culture. The bacterial cells were removed to obtain a supernatant containing the recombinant type I collagen. The supernatant was then separated and purified to obtain the recombinant type I collagen.

8. The preparation method according to claim 7, characterized in that, The separation and purification method is at least one of the following: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

9. The application of the recombinant type I collagen of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, and / or the recombinant strain of claim 5, characterized in that, The application refers to its use in the preparation of cosmetics, health products, medical devices, and / or tissue engineering materials.

10. The application as described in claim 9, characterized in that, The medical devices include medical dressings, medical spray dressings, medical gel dressings, or medical freeze-dried powder dressings; The tissue engineering materials include subcutaneous fillers, artificial bone, artificial skin, oral absorbable biomembranes, bone implants, vascular stents, hemostatic agents, procoagulants, intercellular matrix scaffolds, or collagen sponges.

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