A recombinant humanized type II collagen, its preparation method and application

Abstract

The present invention belongs to the field of biological materials, and particularly relates to a recombinant humanized type II collagen, a preparation method thereof, and an application thereof. The present invention provides a recombinant humanized type II collagen, which is highly expressed through a prokaryotic system, and a recombinant humanized type II collagen with a relatively high purity is obtained after separation and purification. The recombinant humanized type II collagen of the present invention not only has a low preparation cost, but also has the effect of promoting the proliferation of chondrocytes and can be effectively applied to cartilage repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and in particular relates to a recombinant humanized type II collagen and a preparation method and application thereof. Background Art

[0002] Type II collagen is an important member of the collagen family. It is mainly found in cartilage tissue (such as hyaline cartilage, fibrocartilage and elastic cartilage), and is also distributed in small amounts in tissues such as the vitreous body, embryonic cornea, inner ear and optic nerve membrane. Type II collagen has a triple helical structure and is synthesized by chondrocytes. It is mainly in the form of homotrimers ( α 1(II)3). Its synthesis involves the hydroxylation of proline and lysine, and these modifications are crucial for stabilizing the collagen triple helical structure.

[0003] The fibrous network formed by type II collagen gives cartilage tissue good tensile resistance. It provides an environment for chondrocytes to attach and grow, maintaining the spherical morphology and phenotypic stability of the cells. Its adhesion to cells can also activate signaling pathways, regulating cell proliferation, survival and differentiation, and can be widely used in cartilage repair and joint health.

[0004] Although type II collagen scaffolds demonstrate excellent cell guidance and matrix formation capabilities in cartilage repair, the mechanical properties of the regenerated tissue are generally inferior to those of native cartilage. For example, in animal models, cartilage tissue repaired using type II collagen scaffolds exhibits cartilage-like characteristics in terms of cellular structure and matrix composition, but its tensile and compressive properties are still inferior to those of native cartilage. In addition, the extraction and purification costs of type II collagen are relatively high. Therefore, there is a need to find a type II collagen with low production costs and better cartilage repair capabilities. Summary of the Invention

[0005] The main purpose of the present invention is to provide a recombinant humanized type II collagen protein and its preparation method and application. The recombinant humanized type II collagen protein obtained by large-scale production through a prokaryotic expression system has low preparation cost and can promote chondrocyte proliferation and thus promote cartilage repair.

[0006] One aspect of the present invention is to provide a recombinant humanized type II collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] GLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGGPPGPPGPGIDMSAFAGLGPREKGPGPPGERGAPGNRGFPGQDGLAGPKGAPGERGPSGLAGPKGANGDPGRPGEPGRGFTGLQGLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGANGIPGPIGPPGPRGRSGFTYTALKDGCTKHTGKWGKGPAGER;

[0009] One aspect of the present invention is to provide a nucleic acid molecule encoding recombinant humanized type II collagen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] SEQ ID NO.2:

[0011] GGCCTGCCGGGCCCGCCGGGCCCGAGCGGCGATCAGGGCGCGAGCGGCCCGGCGGGCCCGAGCGGCCCGCGCGGCCCGCCGGGCCCGGTGGGCCCGAGCGGCAAAGATGGCGGCCCGCCGGGCCCGCCGGGCCCGGGCATTGATATGAGCGCGTTTGCGGGCCTGGGCCCGCGCGAAAAAGGCCCGGGCCCGCCGGGCGAACGCGGCGCGCCGGGCAACCGCGGCTTTCCGGGCCAGGATGGCCTGGCGGGCCCGAAAGGCGCGCCGGGCGAACGCGGCCCGAGCGGCCTGGCGGGCCCGAAAGGCGCGAACGGCGATCCGGGCCGCCCGGGCGAACCGGGCCGCGGCTTTACCGGCCTGCAGGGCCTGCCGGGCCCGCCGGGCCCGAGCGGCGATCAGGGCGCGAGCGGCCCGGCGGGCCCGAGCGGCCCGCGCGGCCCGCCGGGCCCGGTGGGCCCGAGCGGCAAAGATGGCGCGAACGGCATTCCGGGCCCGATTGGCCCGCCGGGCCCGCGCGGCCGCAGCGGCTTTACCTATACCGCGCTGAAAGATGGCTGCACCAAACATACCGGCAAATGGGGCAAAGGCCCGGCGGGCGAACGC;

[0012] One aspect of the present invention is to provide a vector comprising a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.2.

[0013] One aspect of the present invention is to provide a bacterium comprising the above-mentioned vector. Preferably, the bacterium is Escherichia coli.

[0014] One aspect of the present invention is to provide a method for preparing recombinant humanized type II collagen, specifically:

[0015] (1) Synthesize the gene fragment according to the nucleotide sequence shown in Seq ID NO. 2, ligate the synthesized gene fragment overnight with T4 ligase, and insert it between the restriction sites Nco I and Xho I on the pET32a plasmid to obtain a recombinant plasmid;

[0016] (2) The recombinant plasmid was mixed with DH5α competent cells, electroporated, and then sorbitol solution was added. After mixing, the cells were transferred to a sterile EP tube and incubated at 37°C for 1 hour. The cells were then spread on a sodium ampicillin-LB solid plate and incubated at 37°C for 24 hours until a single colony was grown. A single colony was picked for colony PCR to verify the correctness of the positive clone, and the recombinant prokaryotic expression vector was successfully constructed.

[0017] (3) Pick the correctly expressed plasmid vector from the colony and transform it into Escherichia coli BL21. Use ampicillin sodium-LB solid plate to screen single-spot clones. Add 5 ml of LB medium and culture overnight. Transfer at a ratio of 1:100 and culture in a shake flask at 37°C until the OD600 is between 0.4 and 0.6. Add IPTG at a ratio of 1:5000 and culture at 20°C for 8-12 hours. Collect the cells by centrifugation and save them for the next purification step.

[0018] (4) Wash the pellet with PBS buffer, resuspend the pellet in 20-40 ml volume, lyse the bacteria with lysozyme combined with Triton-X, and disrupt the bacteria by ultrasonication in an ice-water mixture environment, ultrasonication for 2 seconds, 5 seconds interval, ultrasonication for 20 minutes, centrifugation at 12000 rpm for 20 minutes, and collect the supernatant;

[0019] (5) Purifying the supernatant by a cation exchange chromatography column, eluting and collecting the protein corresponding to the elution peak, thereby obtaining recombinant humanized type II collagen with a purity of 97%.

[0020] Another aspect of the present invention provides an application of recombinant humanized type II collagen. Preferably, the application is used to prepare a biomaterial that promotes chondrocyte proliferation and thus promotes cartilage repair.

[0021] Beneficial technical effects of the present invention:

[0022] The present invention constructs a prokaryotic expression system to express recombinant humanized type II collagen, which is ultimately isolated and purified. Electron microscopy reveals a distinct porous, fibrous network structure similar to native type II collagen, suggesting its potential for application in biomedical materials. In vitro cell testing demonstrates its ability to promote chondrocyte proliferation, and in vivo animal experiments demonstrate its ability to promote cartilage repair, suggesting its potential for widespread biomedical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 : Scanning electron microscopy image of recombinant humanized type II collagen.

[0025] Figure 2 : Experiment on promoting proliferation of human chondrocyte C28I2 cells by recombinant humanized type II collagen in vitro.

[0026] Figure 3 : In vivo study on promoting cartilage repair by recombinant humanized type II collagen. DETAILED DESCRIPTION

[0027] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0028] Example 1: Preparation of recombinant humanized type II collagen

[0029] The gene fragment was synthesized according to the nucleotide sequence shown in Seq ID NO.2, and the synthesized gene fragment was ligated overnight with T4 ligase and inserted between the restriction sites NcoⅠ and XhoⅠ on the pET32a plasmid to obtain a recombinant plasmid;

[0030] The recombinant plasmid was mixed with DH5α competent cells, electroporated, and then sorbitol solution was added. After mixing, the cells were transferred to a sterile EP tube and incubated at 37°C for 1 hour. The cells were then spread on a sodium ampicillin-LB solid plate and incubated at 37°C for 24 hours until a single colony was grown. A single colony was picked for colony PCR to verify the correctness of the positive clone, and the recombinant prokaryotic expression vector was successfully constructed.

[0031] Correctly expressed plasmid vectors were selected from the colonies and transformed into E. coli BL21. Single-spot clones were screened using ampicillin sodium-LB solid plates. 5 ml of LB medium was added and cultured overnight. The cells were transferred at a 1:100 ratio and grown in a shake flask at 37°C to an OD600 of 0.6. IPTG was added at a 1:5000 ratio and cultured at 20°C for 12 hours. The cells were collected by centrifugation and stored for purification.

[0032] The pellet was washed with PBS buffer and resuspended in 30 ml of PBS buffer. The bacteria were lysed with lysozyme and Triton-X. The cells were then sonicated in an ice-water mixture for 2 s with a 5 s pause for 20 min. The pellet was centrifuged at 12,000 rpm for 20 min and the supernatant was collected.

[0033] The induced supernatant was purified by a cation exchange chromatography column, and the protein corresponding to the elution peak was eluted and collected to obtain recombinant humanized type II collagen with a purity of 97%.

[0034] Example 2: Scanning electron microscopy of recombinant humanized type II collagen

[0035] (1) Sample preparation: Apply conductive glue on the sample table, gently take out the freeze-dried product, stick it on the conductive glue, and perform gold vaporization.

[0036] (2) Observation: The surface structure of the freeze-dried collagen product was observed at a magnification of 100 using an electron scanning microscope.

[0037] Scanning electron microscopy results of recombinant human type II collagen are as follows Figure 1 As shown. Figure 1 It can be seen that the sample has an obvious porous fiber network structure, which indicates that it has the potential to be applied in the field of biomedical materials.

[0038] Example 3: In vitro cell experiment on promoting chondrocyte proliferation by recombinant humanized type II collagen

[0039] The recombinant humanized type II collagen of Example 1 was used to promote cell proliferation. The cells used were human chondrocytes C28I2.

[0040] (1) Human chondrocytes C28I2 were digested, centrifuged, resuspended, and diluted to 2×104 cells / mL. 100 μL was inoculated into each well of a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator for 24 h. The 96-well cell culture plate was divided into group A (blank group, no treatment), group B (negative control group, PBS added), group C (experimental group, 0.05 mg / mL recombinant humanized type II collagen added), and group D (positive control group, 0.05 mg / mL type II chicken cartilage collagen added), with 6 replicates per group.

[0041] (2) The culture medium was discarded, and group A was added with culture medium, group B was added with 0.05 mg / mL PBS and culture medium, group C was added with 0.05 mg / mL recombinant humanized type II collagen and culture medium, and group D was added with 0.05 mg / mL type II chicken cartilage collagen and culture medium. The culture was carried out at 37°C, 5% CO2, and the amount of culture medium added was 100 μL. The amount of PBS, type II chicken cartilage collagen, and recombinant humanized type II collagen of Example 1 were all 100 μL.

[0042] (3) Add 20 μL of MTT to each well and incubate in a 37°C, 5% CO2 incubator for 4 h. Add 150 μL of DMSO and incubate on a shaker for 10 min to fully dissolve the crystals. Detect the absorbance at 490 nm using a microplate reader (Table 1).

[0043] The results showed that the added recombinant humanized type II collagen of Example 1 had the same activity in promoting chondrocyte proliferation as natural type II chicken cartilage collagen, and the promoting effect was comparable, indicating that the biological activity of the recombinant humanized type II collagen of the present application was close to that of natural type II collagen and had application value ( Figure 2 ).

[0044] Table 1: Experimental results of promoting the proliferation of human chondrocyte C28I2

[0045]

[0046] Example 4: In vivo animal experiment on promoting cartilage repair with recombinant humanized type II collagen

[0047] Four-week-old SPF C57BL / 6 mice were purchased for the construction of cartilage injury models. There were four groups in total, namely group A (blank group, no treatment), group B (negative control group, PBS added), group C (experimental group, 0.05 mg / mL recombinant humanized type II collagen added) and group D (positive control group, 0.05 mg / mL type II chicken cartilage collagen added), with 3 mice in each group.

[0048] The ends of a 21G needle were cut into an outer cylinder, adjusted to be approximately 27 μm shorter than a 300G needle, and the device was used to cause injury. Before starting the experiment, we measured the cartilage thickness of young mice, and the results showed that the thickness was less than 300 μm. Therefore, we chose a fixed depth of 300 μm for the procedure. Under general anesthesia, the hind limbs were disinfected. A medial parapatellar skin incision slightly less than 1 cm in length was then made using a surgical scalpel. The joint capsule was opened and the patella was dislocated laterally to expose the articular surface of the trochlear groove. The femoral epicondyle was gently fixed with surgical forceps, and the needle end of the above device was used to cause a longitudinal full-thickness injury in the patellar groove. Bleeding at the site of cartilage injury confirmed penetration of the subchondral bone.

[0049] After rinsing with saline to remove debris, the knee joint was dislocated and reduced. Group A received no treatment, while Group B received an injection of 0.3 mL of PBS, Group C received an injection of 0.3 mL of 0.05 mg / mL recombinant humanized type II collagen, and Group D received an injection of 0.3 mL of 0.05 mg / mL type II chicken cartilage collagen. The joint capsule and skin were sutured in separate layers. Postoperatively, the patients were kept warm under light until they recovered from anesthesia.

[0050] The rats were kept normally for 4 weeks after surgery, and protein was extracted from 3 rats in each group to verify the expression of SOX-9, a marker of cartilage repair.

[0051] The results showed that the expression levels of SOX-9 protein in Groups C and D were higher than those in Groups A and B. This indicates that the injection of the recombinant humanized type II collagen of Example 1 repaired cartilage damage to a certain extent, and the repair effect was close to that of natural type II collagen, indicating that it has good clinical application prospects ( Figure 3 ).

[0052] The above descriptions are merely optional embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present description and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A recombinant humanized type II collagen, characterized in that, The amino acid sequence of the recombinant humanized type II collagen is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant humanized type II collagen as claimed in claim 1.

3. The nucleic acid molecule according to claim 2, wherein The sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A carrier, characterized in that, The vector contains the nucleic acid molecule as claimed in claim 2 or 3.

5. A bacterium, characterized in that, The bacterium contains the nucleic acid molecule as claimed in claim 2 or 3 or the vector as claimed in claim 4.

6. The bacterium according to claim 5, characterized in that, The bacterium is Escherichia coli.

7. A method for preparing the recombinant humanized type II collagen according to claim 1, characterized in that, The nucleic acid shown in SEQ ID NO.2 is introduced into a plasmid and then transformed into Escherichia coli. The Escherichia coli is cultured and then induced to express, and finally the recombinant humanized type II collagen is obtained by separation and purification.

8. The preparation method according to claim 7, wherein The method described above includes: (1) Synthesize a gene fragment according to the nucleotide sequence shown in Seq ID NO.2, and ligate the synthesized gene fragment overnight with T4 ligase and insert it between the restriction enzyme sites NcoⅠ and XhoⅠ on the pET32a plasmid to obtain a recombinant plasmid; (2) Mix the recombinant plasmid with DH5α competent cells, perform electroporation, then add a sorbitol solution, mix well, transfer it to a sterile EP tube, incubate it statically at 37°C for 1 h, coat it on an ampicillin sodium-LB solid plate, and then culture it inverted at 37°C for 24 h until single colonies grow. Pick a single colony for colony PCR to verify the correctness of the positive clone, and successfully construct a recombinant prokaryotic expression vector; (3) Pick the plasmid vector with correct expression in the colony, transform it into Escherichia coli BL21, screen single-spot clones with an ampicillin sodium-LB solid plate, add 5 ml of LB medium and culture it overnight, transfer it at a ratio of 1:100, culture it in a shaking flask at 37°C until the OD600 is between 0.4 and 0.6, add IPTG at a ratio of 1:5000, culture it at 20°C for 8 - 12 hours, centrifuge to collect the bacterial cells, and store them for the next purification step; (4) Wash the precipitate with PBS buffer, resuspend the precipitate with a volume of 20 - 40 ml, lyse the bacteria with lysozyme in combination with Triton-X, perform ultrasonic cell disruption in an ice-water mixture, ultrasonicate for 2 s and intermittently for 5 s, ultrasonicate for 20 min, centrifuge at 12000 r / min for 20 min, and collect the supernatant; (5) Purify the supernatant through a cation exchange chromatography column, elute and collect the protein corresponding to the elution peak, and thus obtain the recombinant humanized type II collagen with a purity of 97%.

9. Use of the recombinant humanized type II collagen as claimed in claim 1 in the preparation of a biomaterial for cartilage injury repair.

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