Recombinant humanized II-type collagen as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological materials, and particularly relates to recombinant humanized II-type collagen as well as a preparation method and application thereof. The invention provides a recombinant humanized II-type collagen, which is efficiently expressed through a prokaryotic system, and is separated and purified to obtain the recombinant humanized II-type collagen with higher purity. The recombinant humanized II-type collagen is low in preparation cost, has the effect of promoting cartilage cell proliferation, and can be effectively applied to cartilage repair.

Description

Technical Field

[0001] The 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 vitreous body, embryonic cornea, inner ear and optic nerve membrane. Type II collagen has a triple helical structure and is synthesized by chondrocytes, mainly in the form of homotrimers ( α 1(II) 3 Its synthesis involves the hydroxylation of proline and lysine, and these modifications are essential for stabilizing the triple helical structure of collagen.

[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, maintains the spherical morphology and phenotypic stability of the cells, and its adhesion to cells can activate signal pathways, regulate cell proliferation, survival and differentiation, and can be widely used in cartilage repair and joint health.

[0004] Although type II collagen scaffolds show good cell guidance and matrix formation capabilities in cartilage repair, the mechanical properties of the regenerated tissue are generally lower than those of natural cartilage. For example, in animal models, although cartilage tissue repaired using type II collagen scaffolds showed cartilage-like characteristics in cell structure and matrix composition, its tensile and compressive properties were still inferior to those of natural cartilage. In addition, the extraction and purification costs of type II collagen are relatively high. Therefore, it is necessary to find a type II collagen with low preparation cost and better cartilage repair ability. Summary of the invention

[0005] The main purpose of the present invention is to provide a recombinant humanized type II collagen and a preparation method and application thereof. The recombinant humanized type II collagen obtained by large-scale production through a prokaryotic expression system has a low preparation cost and can promote chondrocyte proliferation, thereby promoting 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: GLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGGPPGPPGPGIDMSAFAGLGPREKGPGPPGERGAPGNRGFPGQDGLAGPKGAPGERGSGLAGPKGANGDPGRPGEPGRGFTGLQGLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGANGIPGPIGPPGPRGRSGFTYTALKDGCTKHTGKWGKGPAGER; 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.

[0008] SEQ ID NO.2: ; 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.

[0009] One aspect of the present invention is to provide a bacterium, wherein the bacterium comprises the above-mentioned vector. Preferably, the bacterium is Escherichia coli.

[0010] One aspect of the present invention is to provide a method for preparing recombinant humanized type II collagen, specifically: (1) Synthesizing a gene fragment according to the nucleotide sequence shown in Seq ID NO.2, and ligating the synthesized gene fragment overnight with T4 ligase, and inserting it between the restriction sites NcoⅠ and XhoⅠ on the pET32a plasmid to obtain a recombinant plasmid; (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. After incubation at 37° C. for 1 h, the cells were spread on a sodium ampicillin-LB solid plate, and then incubated at 37° C. for 24 h 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; (3) Pick the correctly expressed plasmid vector from the colony, transform it into Escherichia coli BL21, screen single-spot clones using ampicillin sodium-LB solid plates, add 5 ml LB medium and culture overnight, transfer at a ratio of 1:100, culture in a shake flask at 37°C until OD600 is between 0.4 and 0.6, add IPTG at a ratio of 1:5000, culture at 20°C for 8-12 hours, collect the cells by centrifugation, and store them for the next step of purification; (4) Wash the precipitate with PBS buffer, resuspend the precipitate in a volume of 20-40 ml, lyse the bacteria with lysozyme and Triton-X, and perform ultrasonic lysis in an ice-water mixture environment, ultrasonicating for 2 seconds, with a 5-second interval, ultrasonicating for 20 minutes, centrifuging at 12,000 rpm for 20 minutes, and collecting the supernatant; (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%.

[0011] 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.

[0012] Beneficial technical effects of the present invention: The present invention expresses recombinant humanized type II collagen by constructing a prokaryotic expression system, and finally obtains recombinant humanized type II collagen by separation and purification. The electron microscopy results show that it has an obvious porous fiber mesh structure similar to natural type II collagen, and such a structure indicates that it has the potential to be applied in the field of biomedical materials. Through in vitro cell tests, it is proved that it has the effect of promoting chondrocyte proliferation, and through in vivo animal experiments, it is proved that it has the effect of promoting cartilage repair, and can be widely used in biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 paying creative work.

[0014] Figure 1 : Scanning electron microscope image of recombinant humanized type II collagen.

[0015] Figure 2 : Experiment on promoting proliferation of human chondrocyte C28I2 cells by recombinant humanized type II collagen in vitro.

[0016] Figure 3 : In vivo study on promoting cartilage repair by recombinant humanized type II collagen. DETAILED DESCRIPTION

[0017] 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 claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Example 1: Preparation of recombinant humanized type II collagen The gene fragments were synthesized according to the nucleotide sequence shown in Seq ID NO.2, and the synthesized gene fragments were ligated overnight by T4 ligase and inserted between the restriction sites NcoⅠ and XhoⅠ on the pET32a plasmid to obtain a recombinant plasmid; The recombinant plasmid was mixed with DH5α competent cells, electroporated, and then sorbitol solution was added. After mixing, the mixture was transferred to a sterile EP tube, incubated at 37° C. for 1 hour, and then coated on a sodium ampicillin-LB solid plate. The plate was then 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. Pick the correctly expressed plasmid vector from the colony, transform it into E. coli BL21, screen single-spot clones with ampicillin sodium-LB solid plates, add 5 ml LB medium and culture overnight, transfer at a ratio of 1:100, culture in a shake flask at 37°C until OD600 is 0.6, add IPTG at a ratio of 1:5000, culture at 20°C for 12 hours, collect the cells by centrifugation, and store for the next step of purification; Wash the precipitate with PBS buffer, resuspend the precipitate in 30 ml volume, lyse the bacteria with lysozyme and Triton-X, and then perform ultrasonic lysis in an ice-water mixture environment, ultrasonicate for 2 seconds, rest for 5 seconds, ultrasonicate for 20 minutes, centrifuge at 12000 r / min for 20 minutes, and collect the supernatant; The induced supernatant is purified by a cation exchange chromatography column, and the protein corresponding to the elution peak is eluted and collected to obtain recombinant humanized type II collagen with a purity of 97%.

[0019] Example 2: Scanning electron microscopy of recombinant humanized type II collagen (1) Sample preparation: Stick conductive glue on the sample table, gently take out the freeze-dried product, stick it on the conductive glue, and perform gold vaporization treatment.

[0020] (2) Observation: The surface structure of the freeze-dried collagen was observed at a magnification of 100 using an electron scanning microscope.

[0021] The results of scanning electron microscopy observation 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.

[0022] Example 3: In vitro cell experiment on promoting chondrocyte proliferation by recombinant humanized type II collagen The recombinant humanized type II collagen of Example 1 was used to promote cell proliferation. The cells used were human chondrocytes C28I2.

[0023] (1) Human chondrocytes C28I2 were digested, centrifuged, resuspended, and diluted to 2×10 4 100 μL was inoculated into each well of a 96-well cell culture plate and incubated at 37°C with 5% CO 2 Cultured in an incubator for 24 h; 96-well cell culture plates were 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 replicate wells in each group; (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 cells were incubated at 37°C with 5% CO. 2 , cultured for 48 hours, the amount of culture medium added was 100 μL, and the amount of PBS, type II chicken cartilage collagen, and recombinant humanized type II collagen of Example 1 added were all 100 μL; (3) Add 20 μL MTT to each well and incubate at 37°C with 5% CO 2 Incubate in an incubator for 4 h. Add 150 μL DMSO and incubate on a shaker for 10 min to fully dissolve the crystals. Detect the absorbance at 490 nm using an ELISA reader (Table 1).

[0024] The results showed that the added recombinant humanized type II collagen of Example 1 had the same activity in promoting chondrocyte proliferation as the 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 the natural type II collagen, and had application value ( Figure 2 ).

[0025] Table 1: Experimental results of promoting proliferation of human chondrocyte C28I2

[0026] Example 4: In vivo animal experiment on promoting cartilage repair with recombinant humanized type II collagen SPF-grade 4-week-old C57BL / 6 mice were purchased for the construction of cartilage injury model. There were 4 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.

[0027] The two 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 create the 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 to proceed. 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 create a longitudinal full-thickness injury in the patellar groove. Bleeding at the site of cartilage injury confirmed penetration of the subchondral bone.

[0028] After flushing with saline to remove debris, the knee joint was dislocated and reduced. Group A did not receive any treatment, group B was injected with 0.3 mL of PBS, group C was injected with 0.3 mL of 0.05 mg / mL recombinant humanized type II collagen, and group D was injected with 0.3 mL of 0.05 mg / mL type II chicken cartilage collagen. The joint capsule and skin were sutured in different layers. After surgery, they were given light warmth until they recovered from anesthesia.

[0029] The rats were fed 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 protein for cartilage repair.

[0030] The results showed that the expression of SOX-9 protein in group C and group D was higher than that in group A and group B. It can be seen that the cartilage damage was repaired to a certain extent by injecting the recombinant humanized type II collagen of Example 1, and the repair effect was close to that of natural type II collagen, which has a good clinical application prospect ( Figure 3 ).

[0031] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention's specification and drawings, or directly / indirectly applied 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 according to claim 1.

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

2.

4. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 2 or 3.

5. A bacterium, characterized in that The bacterium comprises the nucleic acid molecule of claim 2 or 3 or the vector of claim 4.

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

7. A method for preparing 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 recombinant humanized type II collagen is obtained by separation and purification.

8. The preparation method according to claim 7, characterized in that: The method comprises: (1) Synthesizing a gene fragment according to the nucleotide sequence shown in Seq ID NO.2, and ligating the synthesized gene fragment overnight with T4 ligase, and inserting it between the restriction sites NcoⅠ and XhoⅠ on the pET32a plasmid to obtain a recombinant plasmid; (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. After incubation at 37° C. for 1 h, the cells were spread on a sodium ampicillin-LB solid plate, and then incubated at 37° C. for 24 h 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; (3) Pick the correctly expressed plasmid vector from the colony, transform it into Escherichia coli BL21, screen single-spot clones using ampicillin sodium-LB solid plates, add 5 ml LB medium and culture overnight, transfer at a ratio of 1:100, culture in a shake flask at 37°C until OD600 is between 0.4 and 0.6, add IPTG at a ratio of 1:5000, culture at 20°C for 8-12 hours, collect the cells by centrifugation, and store them for the next step of purification; (4) Wash the precipitate with PBS buffer, resuspend the precipitate in a volume of 20-40 ml, lyse the bacteria with lysozyme and Triton-X, and perform ultrasonic lysis in an ice-water mixture environment, ultrasonicating for 2 seconds, with a 5-second interval, ultrasonicating for 20 minutes, centrifuging at 12,000 rpm for 20 minutes, and collecting the supernatant; (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%.

9. Use of the recombinant humanized type II collagen according to claim 1 in preparing a biomaterial for promoting chondrocyte proliferation.

Images