A recombinant human type III collagen, its preparation method and application

Abstract

This invention discloses a recombinant human type III collagen, its preparation method, and its applications. The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO.1. This invention optimizes the codons for the CHO cell expression system (as shown in SEQ ID NO.2), successfully achieving efficient expression of the recombinant human type III collagen. Furthermore, since CHO cells, as mammalian cells, possess the ability to fold and modify proteins, this facilitates the formation of positive glycosylation modification of the product. Experimental verification shows that the recombinant human type III collagen prepared by this invention exhibits activity in promoting the adhesion, proliferation, and migration of human fibroblasts.

Description

Technical Field

[0001] This invention relates to the field of optimized coding gene technology, and in particular to a recombinant human type III collagen, its preparation method, and its application. Background Technology

[0002] Type III collagen is a key component of the extracellular matrix, especially in soft tissues such as skin and blood vessels, where it exists in a fine network structure, giving tissue elasticity and resilience, and playing an irreplaceable regulatory role in early wound healing, cell migration, and proliferation. However, naturally occurring type III collagen is extremely limited and poses risks of animal-derived pathogens and immunogenicity issues.

[0003] In existing technologies, recombinant human type III collagen is mainly expressed in microbial systems (such as *E. coli*), but this method suffers from the following fundamental drawbacks: The prokaryotic system lacks endoplasmic reticulum-modifying enzymes found in mammalian cells, resulting in insufficient hydroxylation of proline and lysine in the expressed collagen, preventing the formation of a stable triple helix structure, leading to poor thermal stability and low biological activity. It often exists as inclusion bodies, requiring complex renaturation processes, resulting in low yields and a tendency to form misaggregates. Natural type III collagen contains specific glycosylation sites, which significantly influence its protein stability and cell-cell interactions, and these sites cannot be achieved in the prokaryotic system.

[0004] Therefore, developing a recombinant human type III collagen that can be solublely expressed in CHO cells and has a stable structure and excellent activity has enormous market potential and clinical value. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a recombinant human type III collagen, its preparation method, and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a recombinant human type III collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The second aspect is to provide a nucleic acid molecule that encodes the recombinant human type III collagen described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The third aspect is to provide a recombinant expression vector containing the nucleic acid molecule described in the second aspect.

[0009] The fourth aspect is to provide a host cell containing the recombinant expression vector described in the third aspect.

[0010] Furthermore, the host cell is a CHO cell.

[0011] The fifth aspect is to provide a method for preparing the aforementioned recombinant human type III collagen, specifically including the following steps: (1) Protein sequence design: Based on human type III collagen, an amino acid sequence as shown in SEQ ID NO.1 was designed; (2) Gene design and synthesis: Design the nucleic acid sequence according to the amino acid sequence described in step (1), optimize the codons, and synthesize the nucleic acid fragment encoding the recombinant human type III collagen. (3) Construction of expression vector: The nucleic acid fragment synthesized in step (2) is ligated into the expression vector to obtain the recombinant expression vector; (4) Cell culture and transfection: The recombinant expression vector from step (3) is transfected into host cells for culture; (5) Purification: Recombinant human type III collagen was isolated and purified from the culture.

[0012] Further, the expression vector in step (3) is pCDNA3.4 plasmid.

[0013] Furthermore, the host cell in step (4) is a CHO cell.

[0014] The sixth aspect is to provide the use of the aforementioned recombinant human type III collagen in the preparation of tissue repair products.

[0015] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention optimizes the codons of the CHO cell expression system (as shown in SEQ ID NO.2), successfully achieving efficient expression of recombinant human type III collagen. Furthermore, since CHO cells, as mammalian cells, possess the ability to fold and modify proteins, this facilitates the formation of positive glycosylation modifications in the product. Experimental verification shows that the recombinant human type III collagen prepared by this invention exhibits activity in promoting the adhesion, proliferation, and migration of human fibroblasts. Attached Figure Description

[0016] Figure 1 This is a gene expression vector map of the recombinant human type III collagen (rhCol III) of this invention.

[0017] Figure 2 This is an SDS-PAGE electrophoresis analysis of recombinant human type III collagen in Example 1.

[0018] Figure 3 The results of recombinant human type III collagen adhesion to human fibroblasts in Example 2 are shown, with (left) recombinant human type III collagen and (right) blank control.

[0019] Figure 4 The results show the detection of the proliferative activity of recombinant human type III collagen on human fibroblasts in Example 3.

[0020] Figure 5 The results of the detection of human fibroblast migration by recombinant human type III collagen in Example 4 are shown. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0022] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0023] Example 1 (1) Gene design and synthesis Based on the sequence characteristics of human type III collagen, a collagen sequence was designed, the amino acid sequence of which is shown in SEQ ID NO. 1, consisting of a tandem repeat of amino acids 180-333 of the α1 chain, repeated 4 times. The tail contains a His-Tag of 6 histidine residues for identification and purification. The amino acid sequence of this recombinant human type III collagen fragment is shown below: SEQ ID NO. 1 MGWSCIILFLVATATGVHSGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGA RGNDGARGSDGQPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGA RGSDGQPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQ PGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPHHHHHH The encoding nucleic acid sequence was reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host CHOx cell line. After optimization, the corresponding nucleic acid fragment encoding recombinant human type III collagen (SEQ ID NO. 2) was obtained.

[0024] SEQ ID NO. 2:

[0025] Gene synthesis: Based on the nucleic acid sequence, the nucleic acid fragment encoding the recombinant human type III collagen fragment was synthesized by Cysofa Biotechnology Co., Ltd.

[0026] (2) Construction of expression vector pCDNA3.4-III The obtained nucleic acid fragments were ligated to the pCDNA3.4 plasmid through multiple cloning sites (XbaI and XhoI) to obtain the recombinant plasmid pCDNA3.4-III.

[0027] (3) Culture of CHO cells 1) Cell resuscitation: One tube of CHO cells (1 mL) frozen in liquid nitrogen was rapidly thawed by shaking in a 37°C water bath. The thawed cells were transferred to a 50 mL centrifuge tube, 10 mL of serum-free culture medium was added and mixed well, centrifuged at 400 g for 5 min, the supernatant was discarded, 20 mL of serum-free culture medium was added and mixed well, and then all cell suspensions were added to a culture shake flask and cultured in a 37°C, 5% CO2 110 rpm incubator. The serum-free culture medium was purchased from Maibang Biotechnology Co., Ltd.

[0028] 2) Cell passage: Once the cell density reaches 4E6 or higher, passage culture can be performed. After the first passage, culture the cells until they are in good condition and have a viability of over 90%. Take cells in the logarithmic growth phase and dilute them to 0.5 × 10⁶. 6 Cells per mL were seeded into 250 mL shake flasks and cultured in an incubator at 37°C and 5% CO2 at 110 rpm. Transfection was performed when the cell density reached 4E6.

[0029] (4) Recombinant expression vector transfected CHO cells Cell preparation before transfection: Two days before transfection, cells were inoculated at 0.5 × 10⁻⁶ cells per cell line. 6 Seeds were taken at 100 cells / mL, with a working volume of 27mL. After 2 days of culture, the density reached (4~5)×10⁻⁶ cells / mL on the same day. 6 A cell / mL count with a viability >98%, normal cell morphology, and no clumping is required for instantaneous transfer.

[0030] Preparation of the transfection complex: a) Dilute 60 μg of plasmid to 1.5 mL with Transbuffer A diluent, mix well, and incubate at room temperature for 5-10 min; b) Dilute 180 μg of PEI to 1.5 mL with Transbuffer A diluent, mix well, and incubate at room temperature for 5-10 min; c) Slowly add the PEI diluent to the plasmid diluent, mix well, and incubate at room temperature for 5 min, not exceeding 10 min; d) Add the plasmid-PEI complex dropwise to the shake flask, mixing well as you add, and return it to the shaker for further incubation.

[0031] Post-transfection treatment: 18-24 hours after transfection, add 2% (e.g., working volume 30mL * 2% = 0.6mL) of enhancer TiterMaxerA, along with 3% feed medium MaxFX and 0.3% feed medium MaxFB, and incubate at 33℃ in a shaker with 5% CO2 concentration.

[0032] In the later stages, every other day (D3, D5, D7, D9, D11, D13), 3% MaxFX feed medium and 0.3% MaxFB feed medium were added.

[0033] Ensure that the glucose content is not lower than 1 g / L during the culture process (measure the glucose on the feeding day before D7, if it is lower than 4 g / L, supplement to 8 g / L, measure the glucose every day after D7, and select the upper limit of glucose supplementation according to the actual metabolic status of the cells), and harvest after transfection when the cell viability is lower than 70%.

[0034] (5) Harvesting and purification of recombinant human type III collagen 1) Separation of cells and supernatant: When cell culture is finished, first transfer the culture medium to a centrifuge tube and centrifuge at low speed (e.g., 300-500×g, 5-10 minutes) at 4°C to precipitate the cells and collect the supernatant containing the target protein.

[0035] Note: The operation should be performed at a low temperature as much as possible to slow down the activity of the protease.

[0036] 2) Clarification of the supernatant: To completely remove residual cells, cell debris, and large particles and avoid clogging the purification medium, the supernatant after centrifugation needs to be microfiltered. Use a 0.22μm or 0.45μm filter membrane for vacuum filtration or a syringe filter.

[0037] 3) Chromatography column (using gravity or FPLC systems such as ÄKTA): Equilibrate pre-packed columns such as HisTrap with binding buffer (e.g., 20mM sodium phosphate, 500mM NaCl, pH 7.4); Sample loading: Pass the clarified sample through the chromatography column at an appropriate flow rate, and the target protein binds to the column. Washing: Rinse the column with wash buffer containing a low concentration of imidazole (e.g., 10-40mM) to remove non-specifically bound contaminating proteins. Elution: Competitively elute the target protein with elution buffer containing a high concentration of imidazole (e.g., 250-500mM).

[0038] (6) SDS-PAGE protein electrophoresis detection Sample preparation: Collect the purified sample, add loading buffer and mix well, boil in water for 10 minutes, cool naturally, and set aside.

[0039] Electrophoresis: Use GenScript SurePAGE™ precast gel (4-12%) for loading, and electrophoresis at 140V for 45-55 minutes until the bromophenol blue band reaches the bottom of the gel.

[0040] Coomassie Brilliant Blue R-250 staining (microwave oven method): 1) Prepare the staining solution: Dissolve Coomassie Brilliant Blue R250 to a final concentration of 0.1% (w / v) in a mixture of 40% ethanol and 10% acetic acid. 2) Prepare the destaining solution: Dissolve 10% (v / v) ethanol and 7.5% (v / v) acetic acid together. 3) After electrophoresis, pry open the gel plate and remove the gel, then place it in a staining container containing 100 mL of staining solution. 4) Cover the container and microwave on high for 8 minutes. To avoid danger, do not allow the solution to boil. 5) Remove the staining container from the microwave and gently shake it on a destaining shaker at room temperature for 5 minutes. 6) Discard the staining solution and carefully wash the gel with deionized water. 7) Discard the deionized water and add 100 mL of destaining solution. 8) Cover the container and microwave on high for 8 minutes. 9) Discard the decolorizing solution, add new decolorizing solution, and repeat step 8. 10) Remove from the microwave oven and place on a decolorizing shaker at room temperature, gently shaking until the background is clear.

[0041] Experimental results are as follows Figure 2 As shown, recombinant human type III collagen was expressed, and the target protein showed almost no degradation bands after purification.

[0042] Example 2: Adhesion of recombinant human type III collagen to fibroblasts The recombinant human type III collagen prepared in Example 1 was diluted to 0.1 mg / mL with PBS. Using untreated TC-treated 96-well cell culture plates, 100 μL of the above dilution was added to each well (an equal volume of PBS was added to the blank control wells), and the plates were incubated at 4°C for 24 h for coating. Human fibroblasts (HSF) cultured to approximately 70%–80% confluence were then cultured in high-glucose DMEM medium to a cell density of 1 × 10⁻⁶ cells / well. 5 Cell suspension was prepared at 100 μL / mL. The coating solution in the 96-well plate was discarded, and 100 μL of cell suspension was added to each well. After incubation for 5 h, the cell adhesion effect was observed using an inverted microscope.

[0043] like Figure 3 As shown, at a protein concentration of 0.1 mg / mL, the cells in the recombinant human type III collagen treatment group prepared in this invention exhibited good cell adhesion. Compared with the blank control, the cells were able to adhere very well and fully extend into a fibroblast morphology, showing significantly stronger cell adhesion activity.

[0044] Example 3 The recombinant human type III collagen prepared in Example 1 was used to detect its effect on the proliferation of HSF human skin fibroblasts. The specific steps are as follows: Cell seeding: Human HSF cells (purchased from ATCC cell bank, product catalog number HTX2132) were seeded into sterile 96-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 10% in 96-well plates (DMEM medium containing 10% FBS) and cultured for 24 hours.

[0045] Cell treatment: Discard the culture medium and wash three times with PBS. Experimental group: Add 100 μL of recombinant human type III collagen solution (diluted with DMEM, concentration 0.1 mg / mL). Control group (CK group): Add an equal volume of DMEM medium (without collagen). A blank well (cell-free, containing only DMEM medium) was also provided for background subtraction. Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 24 hours.

[0046] CCK-8 assay: After incubation, discard the supernatant, add 100 μL of 10% CCK-8 solution (serum-free DMEM) to each well, and then incubate in a cell culture incubator for 2 hours.

[0047] Absorbance measurement: After removing the plate, gently shake it and pipette 100 μL of the test solution into a new ELISA plate. Measure the absorbance (OD) at 450 nm using a multi-mode microplate reader. Record the absorbance of the experimental wells as At, the absorbance of the control wells as Ac, and the absorbance of the blank wells as A0. Cell viability = (At - A0) / (Ac - A0) × 100%.

[0048] like Figure 4 As shown, the recombinant human type III collagen prepared by this invention has a significant effect on promoting cell proliferation.

[0049] Example 4 The effect of recombinant human type III collagen on promoting HSF cell migration was detected using a cell scratch assay. The specific steps are as follows: Mark each well on the back of the 24-well plate with three equal divisions both horizontally and vertically using a marker pen. Each well should be approximately 5×10 cm². 5 HSF cells were cultured. When the cells reached 95% confluence, a 10µL pipette tip was used to create cell scratches. The cells were washed three times with PBS to remove the scratched cells. The area of ​​the scratch at the intersection of the horizontal and vertical scratches was photographed under a microscope and recorded as the 0h scratch area. Culture medium was added to the blank control group, while recombinant human type III collagen solution (diluted with DMEM, concentration 1 mg / mL) was added to the sample group, and culture continued. Photographs were taken under a microscope after 5h and 25h of culture.

[0050] The results are as follows Figure 5 As shown, the recombinant human type III collagen prepared in this invention can significantly promote the migration of HSF cells, and the scratch healing speed is significantly faster than that of the blank control group, indicating that it has good cell migration promotion properties.

[0051] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

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

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant human type III collagen of claim 1, and its nucleotide sequence is shown in SEQ ID NO.

2.

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

4. A host cell, characterized in that, The host cell contains the recombinant expression vector as described in claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a CHO cell.

6. A method for preparing recombinant human type III collagen as described in claim 1, characterized in that, Specifically, the steps include the following: (1) Protein sequence design: Based on human type III collagen, an amino acid sequence as shown in SEQ ID NO.1 was designed; (2) Gene design and synthesis: Design the nucleic acid sequence according to the amino acid sequence described in step (1), optimize the codons, and synthesize the nucleic acid fragment encoding the recombinant human type III collagen. (3) Construction of expression vector: The nucleic acid fragment synthesized in step (2) is ligated into the expression vector to obtain the recombinant expression vector; (4) Cell culture and transfection: The recombinant expression vector from step (3) is transfected into host cells for culture; (5) Purification: Recombinant human type I collagen was isolated and purified from the culture.

7. The preparation method according to claim 6, characterized in that, The expression vector in step (3) is pCDNA3.4 plasmid.

8. The preparation method according to claim 6, characterized in that, The host cell in step (4) is a CHO cell.

9. The use of the recombinant human type III collagen as described in claim 1 in the preparation of tissue repair products.

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