A recombinant type I collagen, its preparation method and application

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant type I collagen, its preparation method, and its applications. Its amino acid sequence is shown in SEQ ID NO.1. The recombinant type I collagen of this invention is based on a short amino acid sequence from natural type I collagen, with the integrin recognition sequence GFPGER added as a repeating unit, repeated multiple times, with a repetition count of 6. The recombinant type I collagen exhibits extremely good hydrophilicity and stability, high adhesion, and does not cause immune rejection when applied to the human body. It can be used in the preparation of tissue engineering materials such as bone implants and bone repair scaffolds.

Description

Technical Field

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

[0002] As one of the most important human organs, bone is composed of a specific extracellular matrix (ECM), which provides an organic and inorganic composite framework for osteoblasts (OBs) and osteoclasts (OCs), supporting their mechanical properties and cell adhesion surfaces. Bone injury is a common clinical condition, mainly classified into injuries caused by external forces (such as impact) and injuries caused by pathological factors (such as bone infection). Repairing bone defects is a significant challenge in tissue engineering, especially in areas with insufficient blood supply. Establishing timely blood supply connections with the host body is crucial for the stability of tissue-engineered bone (TEB).

[0003] Bone is a complex tissue comprising both organic and inorganic components, providing structural support and contributing to the regulation of mineral homeostasis. Type I collagen is the major structural component of mammalian bone, accounting for 90% of the organic components of the extracellular matrix (ECM). In addition, there are numerous non-collagenous matrix proteins, at least two types of glycoproteins (disaccharides and core proteoglycans), and some minor collagens (primarily type III and type X). Type I collagen is one of the most abundant proteins, widely expressed in connective tissue and the interstitial membrane, and is also a major organic component of bone tissue. The appropriate protein sequence of type I collagen plays an indispensable role in the mechanical strength of bone.

[0004] Currently, almost all natural type I collagen used in biomedical and pharmaceutical applications, as well as tissue-engineered medical products (TEMPs), is primarily animal-derived, with bovine collagen dominating in both quality and quantity. However, these natural type I collagens suffer from low adhesiveness. Summary of the Invention

[0005] To address the above problems, this invention provides a recombinant type I collagen, its preparation method, and its application.

[0006] This invention is achieved through the following technical solution: A recombinant type I collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] A gene encoding the recombinant type I collagen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] An expression vector comprising a nucleotide sequence as shown in SEQ ID NO.2, and the nucleotide sequence as shown in SEQ ID NO.3.

[0009] A host cell containing the expression vector.

[0010] Preferably, the host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0011] A method for preparing recombinant type I collagen involves culturing host cells in a culture medium, inducing the expression of recombinant type I collagen, and then purifying the cells to obtain recombinant type I collagen.

[0012] Preferably, the purification method includes any one of salting out, ultrafiltration, affinity chromatography, and gel filtration chromatography.

[0013] The application of the recombinant type I collagen in the preparation of exogenous materials for bone repair.

[0014] Compared with the prior art, the present invention has the following beneficial effects: A recombinant type I collagen, the amino acid sequence of which is shown in SEQ ID NO.1. This invention constructs a recombinant type I collagen by replacing the short amino acid sequence of a natural human type I collagen with subsequent repeats as repeating units. The addition of the integrin recognition sequence GFPGER after the sequence enhances the adhesion and angiogenesis-promoting properties of the recombinant collagen. It exhibits excellent hydrophilicity and stability, and its application in the human body does not cause immune rejection. Therefore, it can be used in the preparation of tissue engineering materials such as bone implants and bone repair scaffolds. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a diagram of the purification of recombinant type I collagen according to the present invention; in which lane 1 is a marker; lane 2 is the recombinant collagen recovery solution before elution; lane 3 is the recombinant collagen recovery solution after elution.

[0017] Figure 2 The image shows the crystal violet staining results of the cell adhesion experiment after 24 hours of culture according to the present invention; where A is the control group; B is the BSA negative control group; C is the natural type I collagen positive control group; and D is the recombinant type I collagen administration group.

[0018] Figure 3 The images below show fluorescence micrographs of different concentrations of recombinant type I collagen according to the present invention; wherein, A is the fluorescence micrograph of the control group; B is the fluorescence micrograph of the group with 0.5 mg / mL recombinant type I collagen added; C is the fluorescence micrograph of the group with 1 mg / mL recombinant type I collagen added; D is the fluorescence micrograph of the group with 3 mg / mL recombinant type I collagen added; and E is the fluorescence micrograph of the group with 5 mg / mL recombinant type I collagen added.

[0019] Figure 4 This is a statistical result of the effect of different concentrations of recombinant type I collagen on the proliferation activity of hBMSCs cells. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] The inventive concept of this invention is as follows: The inventors constructed the recombinant collagen by replacing the short amino acid sequence of natural human type I collagen and then repeating it multiple times as a repeating unit. The integrin recognition sequence GFPGER, derived from a segment of amino acid residues on type I collagen, promotes cell adhesion, making it widely applicable in fields such as angiogenesis and bone repair. Therefore, by adding the integrin recognition sequence GFPGER to the end of the sequence, the adhesiveness and angiogenesis-promoting properties of the recombinant collagen are enhanced, thereby producing a recombinant collagen with high adhesiveness.

[0023] This invention provides a recombinant type I collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0024] SEQ ID NO.1: GERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGER GERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGERGERGDLGPQGIAGQRGVVGERGERGERGASGFPGER.

[0025] This invention provides a recombinant type I collagen protein, which features codon optimization for host cell expression, and incorporates signal peptide cleavage sites at both ends during the design process. Eco RI and Kpn The I restriction enzyme site facilitates subsequent gene manipulation. Following the above screening, its nucleotide sequence is shown in SEQ ID NO.2.

[0026] SEQ ID NO.2: .

[0027] The present invention provides an expression vector comprising the nucleotide sequence shown in SEQ ID NO.2 above.

[0028] The vector may contain regulatory sequences, such as transcription and translation start and stop codons, that are specific to the type of host to which the vector is to be introduced, such as bacteria, fungi, plants or animals, and, as appropriate, whether the vector is based on DNA or RNA.

[0029] In one specific embodiment, the expression vector is obtained by linking the nucleotide sequence shown in SEQ ID NO.2 with pPICZ alpha A, and its nucleotide sequence is shown in SEQ ID NO.3.

[0030] SEQ ID NO.3:

[0031] The present invention provides a host cell comprising the above-described nucleotide sequence.

[0032] The host cell refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations.

[0033] In one specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0034] In one specific embodiment, the host cell is Pichia pastoris X-33.

[0035] This invention provides a method for preparing any of the aforementioned recombinant type I collagen, comprising the following steps: The expression was performed using the aforementioned host cells, followed by separation and purification.

[0036] The expression of the host cell refers to the culture of the host cell, and the culture medium and culture conditions are well known to those skilled in the art.

[0037] In one specific embodiment, the host cell is Pichia pastoris, and the specific culture conditions after obtaining the Pichia pastoris genetically engineered strain are as follows: The Pichia pastoris genetically engineered strain was inoculated into BMGY medium and cultured at 30℃ and 220 rpm for 22-24 hours until OD500 was reached. 600 Use 18-20g as the seed culture for the upper tank. After expanding the seed culture, inoculate it into an NBS 415 fermenter with an initial volume of 5L at a 10% inoculation rate. The culture temperature is 28℃-30℃, the pH is 5.0-6.0, and the dissolved oxygen is controlled at 20%-30%. After the glycerol is exhausted, start the glycerol feeding culture. When the cell wet weight reaches more than 180g / L, start the induction culture.

[0038] The present invention does not impose any restrictions on the expression method, which can be determined as needed, for example, the expression can be induced expression, and the inducing agent is methanol.

[0039] In one specific implementation, methanol was added for induction culture, with the induction phase temperature at 28°C and pH at 5.0, and the culture was discharged after 48 hours of induction.

[0040] This application does not impose any restrictions on the methods of separation and purification, which can be determined according to the specific circumstances, such as salting out, ultrafiltration, affinity chromatography and gel filtration chromatography.

[0041] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0042] The reagents and consumables used in this invention are all commercially available products.

[0043] Example 1: Expression of recombinant type I collagen First, the secondary structure of type I collagen was analyzed using bioinformatics software to locate variable or non-critical regions, serving as the basis for engineering modification. Based on this, a new artificial amino acid sequence was designed and constructed. This sequence consists of a repetitive module composed of a short peptide unit from natural type I collagen and an integrin recognition sequence (GFPGER), repeated six times to obtain SEQ ID NO.1. The placement of the GFPGER sequence was confirmed by the above analysis, aiming to improve adhesion while ensuring the overall stability of the recombinant protein structure. To prepare the designed protein, its corresponding coding gene was chemically synthesized. During gene synthesis, codon optimization was performed on the coding sequence to improve its expression efficiency in the selected expression host, Pichia pastoris X-33. The optimized nucleotide sequence used for chemical synthesis is shown in SEQ ID NO.2.

[0044] A suitable expression vector was selected; in this invention, the vector used was pPICZ alpha A. Through... Eco RI and KpnI The synthesized target gene is ligated to the vector via restriction enzyme sites to construct a recombinant expression vector, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0045] The recombinant expression vector was introduced into the expression strain Pichia pastoris X-33 via electroporation, using Zeo + High-copy-ratio positive clones were screened using a gradient method, and positive transformants were selected by PCR. Validated positive transformants were picked up with sterile pipette tips and inoculated into BMGY liquid medium, and cultured for 24 h in a shaking incubator at 30°C and 220 rpm. Subsequently, an appropriate amount of culture was transferred to BMMY medium, and the initial OD was adjusted. 600 The culture was continued at the same temperature and rotation speed for 72 hours, with methanol added every 24 hours to maintain a methanol concentration of 1% in the culture medium. The OD of the bacterial culture was measured every 24 hours. 600 The supernatant was collected by centrifugation, and after sample preparation, SDS-PAGE denaturing gel electrophoresis was performed to analyze the protein expression and obtain the target recombinant type I collagen.

[0046] Example 2: Purification of recombinant type I collagen 1. When the supernatant collected in Example 1 is ultrafiltered to 50% of its initial volume, 5 times the volume of pure water is added, and then the mixture is concentrated by ultrafiltration to 5% of its initial volume to obtain the concentrated supernatant.

[0047] 2. Add 60% of the total volume of the concentrated supernatant to saturated ammonium sulfate, stir at room temperature for 30 min, centrifuge at 9000 rpm for 10 min and collect the precipitate. Dissolve the precipitate in 500 mL of 0.05 M PBS with pH 7.0 and filter through a 0.22 μm filter membrane.

[0048] 3. Prepare equilibration buffers based on the protein's isoelectric point: 20 mmol / L sodium phosphate buffer, pH 6.0, denoted as solution A; prepare an elution buffer (pH 6.0) by mixing 20 mmol / L sodium phosphate and 1.0 mol / L NaCl, denoted as solution B. Dilute the PBS protein solution obtained in the previous step with solution A at a ratio of 10:1 to prepare the loading solution. After filtration, load the solution onto a 25 mL CM-Sepharose cation exchange chromatography column. Equilibrate the column with equilibration buffer before loading. After loading, wash with solution A for two column volumes, then perform gradient elution with 70% solution A and 30% solution B at a flow rate of 2 mL / min. Collect the eluted fractions and analyze them using SDS-PAGE. The results are as follows: Figure 1 Lane 1: marker; Lane 2: recombinant collagen recovery solution before elution; Lane 3: recombinant collagen recovery solution after elution.

[0049] 4. Based on the molecular weight distribution of the proteins obtained after ion exchange chromatography, a Sephadex 200 gel column was selected for further purification of the target protein. AKTA procedure: First, the sample was washed with equilibration buffer (0.01 mol / L PBS and 0.05 mol / L NaCl) until the baseline stabilized. Then, the two protein fractions eluted from the ion exchange column in the previous step were loaded onto a gel filtration chromatography column packed with Superdex 200. Elution was performed with elution buffer at a flow rate of 10 mL / min and UV detection at 215 nm. Finally, the eluent target protein fraction was collected after SDS-PAGE electrophoresis.

[0050] 5. Ultrafiltration desalting: Desalting is performed using a G25 desalting column, which uses 25 mL of G25 packing material. The operation process is similar to that of gel filtration chromatography. Each sample is loaded with 6.5 mL and collected with about 8 mL. Desalting is completed 10 minutes after loading.

[0051] 6. The protein was concentrated to 30% of its initial volume by ultrafiltration, then pre-frozen at -20°C for 4 hours, and then transferred to a vacuum freeze dryer for lyophilization. After 72 hours, the lyophilized protein was collected and stored at 4°C for later use.

[0052] Example 3: Cell adhesion experiment of recombinant type I collagen L929 is commonly used as a cell adhesion assay model. Complete culture medium was used as the control group, BSA (bovine serum albumin) as the negative control, natural type I collagen as the positive control, and recombinant type I collagen as the treatment group. Each well was uniformly coated with 500 μL of each medium into 48-well plates. The coated plates were incubated overnight at 4°C. Subsequently, the wells were blocked with 1% (w / v) heat-denatured BSA at room temperature for 1 hour. After blocking, the wells were washed twice with PBS buffer to prepare coated plates. Well-grown L929 cells were counted using a counting chamber, and the cell concentration was adjusted to an appropriate density, ultimately reaching 2.0 × 10⁶ cells / well. 5 Cells were seeded at a density of [cells / mL] in pre-coated 48-well plates. The plates were then incubated in a CO2 incubator for 4 hours. After incubation, the culture medium was removed, followed by three washes with PBS. Next, the cells were fixed with 4% paraformaldehyde solution for 15 minutes, 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 using an optical microscope. Figure 2 As shown, the number of adherent cells in the recombinant type I collagen administration group was significantly higher than that in other groups, indicating that recombinant type I collagen can effectively improve the adhesion of L929 cells.

[0053] Example 4: Cell proliferation experiment of recombinant type I collagen An in vitro cell proliferation model was established to evaluate the cell viability induced by recombinant collagen. In short, logarithmically growing bone marrow mesenchymal stem cells (hBMSCs) were seeded in 96-well tissue culture plates, achieving a cell density of 510 cells per well. 3 The cells were cultured at 37°C in a 5% CO2 incubator for 1 day. The old culture medium was discarded, and recombinant type I collagen was dissolved sequentially in MEM-α medium at concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, and 5 mg / mL. The solutions were filtered and sterilized, and 100 μL was added to each well of a 96-well plate. In the control group, 100 μL of fresh complete culture medium was added. After 24 hours of incubation, cell proliferation was verified using the CCK8 assay, and absorbance was measured at 450 nm. Simultaneously, the cells after 24 hours of incubation were stained with AO / EB, and their growth status after culture in collagen medium was observed under a fluorescence microscope. Figure 3 and Figure 4 As shown in the figure. The results indicate that the recombinant collagen of the present invention can significantly promote the growth of hMSCs cells, which is consistent with the results observed under light microscopy.

[0054] 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.

[0055] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A recombinant type I collagen, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the recombinant type I collagen as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, It comprises the nucleotide sequence as described in claim 2, the nucleotide sequence being shown in SEQ ID NO.

3.

4. A host cell, characterized in that, It includes the expression vector as described in claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

6. The method for preparing recombinant type I collagen according to claim 1, characterized in that, The host cells as described in claim 4 or claim 5 are cultured in a culture medium, and recombinant type I collagen is induced to express and then purified to obtain recombinant type I collagen.

7. The preparation method according to claim 6, characterized in that, The purification method includes any one of salting out, ultrafiltration, affinity chromatography, and gel filtration chromatography.

8. The application of the recombinant type I collagen according to claim 1 in the preparation of exogenous materials for bone repair.

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