Recombinant II-type collagen with cartilage filling effect and application thereof

Abstract

The invention discloses a recombinant II-type collagen with a cartilage filling effect and application of the recombinant II-type collagen. The amino acid sequence of the recombinant II type collagen is as shown in SEQ ID NO: 1. By intercepting amino acids from the 722nd site to the 955th site of the natural II-type collagen, the recombinant II-type collagen which is easy to produce and has a cartilage filling effect superior to that of the full-length II-type collagen can be obtained.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology. Specifically, this invention relates to a recombinant type II collagen with good cartilage-filling effects and its applications. Background Technology

[0002] The development of recombinant type II collagen cartilage filling technology stems from the core needs in the field of cartilage repair and regeneration in clinical practice and the limitations of traditional filling methods.

[0003] In the fields of medicine and aesthetic medicine, problems such as articular cartilage damage (e.g., cartilage wear caused by osteoarthritis), congenital cartilage hypoplasia (e.g., morphological defects of nasal and ear cartilage), and acquired cartilage defects are common. Cartilage tissue has extremely weak self-regenerative capacity. Traditional solutions such as autologous cartilage transplantation have problems such as large donor site trauma and limited donor volume. Allogeneic cartilage is prone to causing immune rejection, while synthetic polymer filler materials lack bioactivity, are difficult to integrate with autologous cartilage, and have insufficient long-term stability.

[0004] With the development of genetic engineering and biosynthesis technologies, breakthroughs have been achieved in recombinant technology targeting type II collagen, the main functional component of cartilage matrix. Natural type II collagen is extracted from animal cartilage, which has limitations such as scarce sources, low purity, high immunogenicity, and susceptibility to pathogens. Recombinant type II collagen, through humanized gene sequence design (mimicking the human body's own type II collagen gene) and controlled expression using microbial fermentation systems (such as yeast and E. coli), achieves high biocompatibility, low immunogenicity, and batch stability.

[0005] However, the current technical problem is that the recombinant expression yield of full-length type II collagen is low; while the truncated protein of full-length type II collagen is easy to express, its cartilage filling effect is significantly different from that of full-length type II collagen. Summary of the Invention

[0006] In view of the above-mentioned technical problems existing in the prior art, the object of the present invention is to provide a truncated recombinant type II collagen with better cartilage filling effect than full-length type II collagen.

[0007] The inventors conducted in-depth research to solve the above-mentioned technical problems and discovered that by extracting amino acids from positions 722 to 955 of natural type II collagen, recombinant type II collagen with better cartilage filling effect than full-length type II collagen can be obtained.

[0008] That is, the present invention includes: 1. A recombinant type II collagen, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] SEQ ID NO: 1 GRVGPPGSNGNPGPPGPPGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGLAGQRGIVGLPGQRGERGFPGLPGPSGEPGKQGAPGASGDRGPP GPVGPPGLTGPAGEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPGPPGSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFT 2. The nucleic acid of the recombinant type II collagen described in item 1.

[0010] 3. An expression vector comprising the nucleic acid described in item 2.

[0011] 4. A host cell in which the expression vector described in item 3 has been introduced. The host cell is preferably Pichia pastoris.

[0012] 5. A method for producing the recombinant type II collagen of item 1, comprising culturing the host cells of item 4 to express the recombinant type II collagen, and collecting the recombinant type II collagen.

[0013] 6. The use of the recombinant type II collagen described in item 1 in the preparation of cartilage filling materials.

[0014] 7. Use of the recombinant type II collagen described in item 1 in the preparation of medical devices for cartilage filling. Attached Figure Description

[0015] Figure 1 This is an SDS-PAGE electrophoresis image of the recombinant type II collagen prepared in Example 1.

[0016] Figure 2 The images show HPLC chromatograms of the recombinant type II collagen prepared in Example 1 before purification (top) and after purification (bottom).

[0017] Figure 3 A figure showing the results of an experiment involving recombinant type II collagen cartilage filling. Detailed Implementation

[0018] The present patent application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present invention.

[0019] Example 1 1. Construction of yeast expression strains A strain expressing recombinant type II collagen as shown in SEQ ID NO: 1 was constructed. The specific operation was as follows: after optimization according to the codon preference of Pichia pastoris, the corresponding target gene was synthesized through whole-genome synthesis, and double-cut at both ends of the gene. After incubation at 16°C overnight, it was transformed into Top10 competent cells, plated on ampicillin-resistant plates, positive transformation factors were removed, plasmids were extracted, linearized with SacI, and then electroporated into Pichia pastoris GS115 competent cells. Multicopy transformants were screened using G418 resistant plates, which are the recombinant type II collagen expression strains.

[0020] 2. Induced expression of the target protein (1) Pick a single colony of the obtained expression strain and add it to 5 mL of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubate overnight at 30°C and 200 rpm for activation; (2) Inoculate 1% of the culture medium into 100 mL of BMCY medium and incubate at 30°C and 200 rpm until OD. 600 =6.0~9.0; (3) Collect the bacterial cells by centrifugation at 25°C for 6 minutes under a centrifugal force of 1500g, and suspend them in 200mL of BMMY liquid medium to make the initial concentration OD. 600 =1.0. Incubate at 30℃ and 200 rpm: (4) Add methanol every 24 hours to a final concentration of 0.5-1.0% to induce expression; (5) After induction for 72 hours, the culture medium was centrifuged at 12,000 rpm for 2 minutes and the supernatant was collected.

[0021] 3. Purification and preparation of recombinant type II collagen The obtained fermentation supernatant was concentrated by ultrafiltration through a 10kD ultrafiltration membrane, and then separated by an MMC ion exchange column. The eluent was eluted with 0.5mol NaCl solution, collected, desalted, concentrated, and freeze-dried to prepare the recombinant type II collagen.

[0022] Dissolve 10 mg of lyophilized powder in 1 mL of physiological saline, dilute to 1 g / L, and then load onto an SDS-PAGE gel for electrophoresis to confirm molecular weight and protein purity. Electrophoresis results are as follows: Figure 1 As shown in the figure, the results indicate that the constructed expression bacteria can successfully express the target protein.

[0023] 4. Determine the purity of recombinant type II collagen. The purity of the recombinant type II collagen was determined using high-performance liquid chromatography (HPLC). The determination method is as follows: The BioMix SEC-300 column was selected. The wavelength was 230 nm, the injection volume was 20 μL, the mobile phase was (A) 5% acetonitrile + 0.05% trifluoroacetic acid and (B) 90% water, the flow rate was 1.0 mL / min, and the column temperature was set to 15 °C.

[0024] The purity of the target proteins prepared after separation and purification was all above 99%, as shown in the liquid chromatography results. Figure 2 As shown.

[0025] Example 2 This example demonstrates the cell proliferation and differentiation of type II recombinant collagen and full-length type II collagen prepared in Example 1. Proliferation experiment Human fibroblast-HSF cells were cultured to the logarithmic growth phase at a concentration of 6.5 × 10⁻⁶. 3 Inoculate one cell per well into a 96-well plate and incubate at 37°C with 5% CO2. Start the experiment after 24 hours. The experiment was divided into a normal group (negative control), a sample group, and a control group (positive control). The original culture medium was discarded, fresh culture medium was added to the normal group, culture medium containing different concentrations of the aforementioned type II recombinant collagen (0.05 mg / mL, 0.1 mg / mL) was added to the sample group, and culture medium containing 0.05 mg / mL, 0.1 mg / mL of commercially available full-length type II collagen was added to the control group. Each group had 6 replicates, and the culture was carried out at 37°C and 5% CO2. After 24 h, the culture medium was discarded, and culture medium containing MTT was added. After 2 h of culture, the culture medium was removed, DMSO solution was added, and the absorbance value of each well was measured at 490 nm. The proliferation rate of the experimental group relative to the normal group was calculated. The results showed that, compared with the normal group, both the control group and the sample group improved cell viability at all concentrations. Compared with the control group (100%), the cell viability of the sample group at 0.05 mg / mL and 0.1 mg / mL was 105±3.4% and 108±2.85%, respectively, showing a more significant cell proliferation effect (p<0.05).

[0026] Differentiation experiment Human chondrocytes (HCs) in good logarithmic growth phase were collected and cultured in complete medium at a concentration of 3 × 10⁶ cells per well. 5 Inoculate 2 mL per well into 6-well plates, incubate at 37°C with 5% CO2 for 24 h, discard the old culture medium, add drug-containing culture medium and co-culture for 48 h before RNA extraction.

[0027] ①RNA extraction: After co-culturing each sample with human chondrocytes (HCs) for 48 h, the old culture medium was discarded on ice, and the cells were washed three times with PBS to remove serum and enzymes. 1 mL of Trizol reagent was added to each well of a 6-well plate, and the plates were incubated on ice for 20 min. Cells were scraped and collected into 1.5 mL EP tubes, and lysed on ice for 20 min. 200 μL of chloroform was added to each EP tube, and the plates were shaken repeatedly for 15 min, then centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new EP tube, an equal volume of isopropanol was added, and the mixture was gently mixed. The plates were incubated at 4 °C for 1 h, then centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and 1 mL of 75% ethanol was added to wash away the organic solvent. The plates were centrifuged at 12000 rpm for 10 min. The ethanol was dried at room temperature until the RNA precipitate became a clear gel, which was then dissolved in 20 μL of sterile, enzyme-free water.

[0028] ②cDNA synthesis Prepare the following system on ice according to the reverse transcription kit:

[0029] Mix well and centrifuge. Incubate at 65℃ for 5 min, then place on ice. Add the reaction solutions from the table below to the above reaction solution:

[0030] Mix well and centrifuge. Incubate at 60°C for 60 min, then at 85°C for 10 min to inactivate reverse transcriptase. The reverse-transcribed cDNA can be used immediately for subsequent experiments or stored at -20°C.

[0031] When measuring RNA concentration, the A260 / A280 ratio should be between 1.7 and 2.1, and the A230 / A260 ratio should be between 1.9 and 2.3.

[0032] ③ Real-time quantitative PCR (RT-qPCR) Take out the FastStart Universal SYBR Green Master and water, dissolve them, and centrifuge gently to ensure all reagents are recovered into the tube. Carefully pipette the mixture to mix, and store on ice.

[0033] Place the 8-tube PCR reaction assembly or PCR microplate wells on ice. In a single 25 μL PCR reaction system, add the reaction components listed in the table below in sequence, and mix carefully without vortexing. Seal the PCR plate with a transparent PCR tube cap and centrifuge at 1500 g for 1 min.

[0034] Place the reaction tubes / plates into the fluorescence PCR instrument and start the program. Using the housekeeping gene GAPDH as a reference, use 2... -ΔΔCtThe relative expression levels of mRNA were calculated using this method, and the results are shown below: Numerical table of expression levels of cell differentiation markers

[0035] Conclusion: The above data analysis shows that the differentiation effect of recombinant type II collagen is higher than that of commercially available recombinant type II collagen.

[0036] Example 3 New Zealand white rabbits (6 months old, weighing 2.5-3.5 kg) were used to construct a full-thickness articular cartilage defect model of the femoral trochlear surface, with a diameter of 5 mm and a depth of 3 mm. Type II full-length collagen (control group) and recombinant type II collagen (experimental group) were injected, respectively. At week 8 post-surgery, the animals were sacrificed to prepare joint specimens. These specimens will be used for subsequent macroscopic evaluation and micro-computed tomography (Micro-CT) experiments to assess the effectiveness of cartilage repair. Results are as follows: Figure 3 As shown.

[0037] like Figure 3 As shown, the results indicate that the cartilage repair capacity of type II recombinant collagen protein is significantly higher than that of commercially available type II full-length collagen protein.

Claims

1. A recombinant type II collagen, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The nucleic acid encoding the recombinant type II collagen of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell in which the expression vector of claim 3 has been introduced. The host cell is preferably Pichia pastoris.

5. A method for producing the recombinant type II collagen of claim 1, comprising culturing the host cells of claim 4 to express the recombinant type II collagen, and collecting the recombinant type II collagen.

6. Use of the recombinant type II collagen according to claim 1 in the preparation of cartilage filling materials.

7. Use of the recombinant type II collagen of claim 1 in the preparation of a medical device for cartilage filling.

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