Recombinant human type II collagen and its preparation method and application

Abstract

The present invention belongs to the technical field of optimized coding genes, and discloses a recombinant human type II collagen protein and a preparation method and application thereof, and its amino acid sequence is shown in SEQ ID No. 1. The collagen protein provided by the present invention has been experimentally verified to have better performance in promoting the proliferation of bone marrow mesenchymal stem cells, and has no cytotoxicity and high safety, and can be widely used in the fields of medicine, medical devices, biomaterials, tissue engineering, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of optimized coding genes, and specifically relates to a recombinant human type II collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is widely present in animals, with rich content and various types. It has a significant impact on the normal function and damage repair of cells, tissues and even organs. At present, at least 28 types of collagen have been discovered. According to their structural characteristics, collagen is divided into fibroblastic collagen, fibroblast-associated collagen (FACIT college), mesh structure-forming collagen and membrane collagen. Type I, type II and type III collagen are the main fibroblastic collagens. Type I and type III collagen are mainly found in connective tissue, skin and other tissues. Type II collagen is mainly found in cartilage tissue.

[0003] The traditional method of obtaining collagen is to extract it from animal tissues. Animal-derived collagen has problems such as limited raw material sources, risk of virus transmission, and poor batch consistency.

[0004] With the development of genetic engineering technology, people are increasingly turning their research focus to recombinant expression to obtain collagen. Recombinant expression of collagen can effectively avoid the problems existing in animal collagen.

[0005] As early as the early 1970s, Miller isolated a new type of collagen from chicken cartilage tissue, which he called type II collagen. Type II collagen is a homotrimeric superhelical structure composed of three identical α1 (II) chains. In addition to the basic biological functions of collagen, such as hemostasis, biocompatibility, biodegradability, and low immunogenicity, type II collagen also has the functions of maintaining the integrity of cartilage tissue, stimulating chondrocyte growth and redifferentiation, and inducing immune tolerance.

[0006] In clinical medicine, due to the extremely low self-healing ability of cartilage tissue, once damage or lesions occur, it must be repaired or even replaced. However, the repair and regeneration of cartilage has always been a major problem in orthopedics. Based on the biocompatibility, degradability, low immunogenicity, and ability to promote the growth and redifferentiation of chondrocytes of type II collagen, type II collagen has become a very promising scaffold material in cartilage tissue engineering. In 1998, M. Nokelainen et al. from the University of Oulu in Finland constructed an insect strain that expresses human Achilles tendon type II collagen, with an expression level of 50 mg / L. CN 117120466 A discloses a recombinant bovine type II collagen for treatment. There have been many studies and applications related to type I and type III recombinant collagen. However, there are relatively few studies related to the recombinant expression of type II collagen.

[0007] In view of this, it will be of great significance to find a recombinant type II collagen that is biologically active and suitable for industrialization. Summary of the invention

[0008] The purpose of the present invention is to overcome the defects in the prior art, provide a recombinant human type II collagen that can be industrially produced and has the function of promoting the proliferation of bone marrow mesenchymal stem cells, and also provide a preparation method and application thereof.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] In one aspect, the present invention provides a recombinant human type II collagen, wherein the recombinant collagen comprises one or more of the following characteristics:

[0011] (1) Its amino acid sequence is shown in SEQ ID No. 1, which is as follows:

[0012] MHHHHHHGDRGDVGEKGPEGAPGKDGGRGLTGPIGPPGPAGANGEKGEVGPPGPAGSAGARGAPGERGETGPPGPAGFAGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPK GARGAQGPPGATGFPGAAGRVGPPGSNGNPGPPGPPGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGLAGQRGIVGLPGQRGERGFPGLPGPSGEPGKQGAP;

[0013] (2) The recombinant collagen is a protein derived from (1) in which the amino acid sequence in (1) has been substituted, deleted or added with one or more amino acids and has collagen activity.

[0014] As some preferred embodiments of the present invention, the nucleotide sequence of the recombinant human type II collagen comprises one or more of the following features:

[0015] (1) The nucleotide sequence is shown in SEQ ID No. 2, which is as follows:

[0016] .

[0017] (2) Any nucleotide sequence obtained by replacing synonymous codons in (1).

[0018] In another aspect, the present invention provides a vector comprising any one or more of the above-mentioned nucleotide sequences.

[0019] In yet another aspect, the present invention provides a host cell, wherein the host cell contains the above-mentioned vector.

[0020] In another aspect, the present invention provides a method for preparing the above-mentioned recombinant human type II collagen, which specifically comprises the following steps:

[0021] (1) Protein sequence design

[0022] Based on human type II collagen, an amino acid sequence as shown in SEQ No. 1 was designed;

[0023] (2) Gene design and synthesis

[0024] Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human type II collagen described in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type II collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type II collagen fragment;

[0025] (3) Construction of expression vector:

[0026] The expression plasmid was obtained by ligating the plasmid to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites;

[0027] (4) Construction and screening of expression strains:

[0028] The plasmid described in step (3) is transferred into the competent host cell by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain;

[0029] (5) Inducible expression

[0030] Inducing the expression strain obtained in step (4) to express, and collecting the bacterial solution;

[0031] (6) Purification

[0032] The bacterial liquid collected in step (5) is subjected to bacterial cell disruption and centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a recombinant human type II collagen freeze-dried product.

[0033] As some preferred embodiments of the present invention, the host cell is Escherichia coli BL21 (DE3).

[0034] In another aspect, the present invention provides a use of the above-mentioned recombinant human type II collagen in the field of medical devices.

[0035] The beneficial effects of adopting the above technical solution are:

[0036] (1) The sequence selected by the present invention has RGD and GAPGER integrin binding sites and can promote cell proliferation. Experimental verification shows that the present invention has a better ability to promote the proliferation of bone marrow mesenchymal stem cells and has no cytotoxicity.

[0037] (2) The recombinant human type II collagen provided by the present invention has no animal-derived infection source, has high biological safety, and can be widely used in the fields of medicine, medical devices, biomaterials, tissue engineering, cosmetics, etc.

[0038] (3) The method for preparing recombinant human type II collagen of the present invention is suitable for industrial large-scale production, which solves the problem of limited source of type II collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the specific implementation manner of the present invention, the drawings required for describing the specific implementation manner are briefly introduced below.

[0040] Figure 1 The electrophoresis diagram of the fermentation cell sample obtained in Example 4; in the figure: M: Marker, 1: before induction, 2: 12h of induction, the band indicated by the arrow is the target protein band;

[0041] Figure 2 This is a sample electrophoresis diagram of the affinity chromatography process of the recombinant human type II collagen HC72 prepared in Example 5; in the diagram: 1: Marker, 2: whole bacteria, 3: supernatant, 4: precipitate, 5: supernatant filtration, 6: flow-through start, 7: flow-through 300ml, 8: flow-through 700ml, 9: flow-through 1000ml, 10: 20% elution, 11: 50% elution, 12: 100% elution, 13: BSA;

[0042] Figure 3 This is an electrophoresis diagram of a sample of the affinity chromatography process of the recombinant human type II collagen HC72 prepared in Example 5; in the diagram: 1: Marker, 2: affinity chromatography sample, 3: flow-through start, 4: flow-through 300ml, 5: flow-through 600ml, 6: flow-through 900ml, 7: 20% elution, 8: 50% elution, 9: 100% elution;

[0043] Figure 4 This is a statistical diagram of the cytotoxicity experimental data of recombinant human type II collagen HC72 at different concentrations in effect example 1; in the figure: 1: blank control, 2: 0.1 mg / ml, 3: 0.5 mg / ml, 4: 1 mg / ml, 5: 2 mg / ml, 6: 5 mg / ml, 7: 10 mg / ml;

[0044] Figure 5 This is a statistical chart of the data of the cell proliferation promotion test of recombinant human type II collagen HC72 in effect example 2; in the figure: 1: blank control, 2: HC72 treatment group, 3: bovine type II collagen.

[0045] Figure 6This is the SDS-PAGE gel electrophoresis diagram of the stability study of recombinant human type II collagen HC72 in effect example 3; in the figure: M: Marker, 1: 0 month, 2: 1 month, 3: 3 months, 4: 6 months, 5: 12 months. DETAILED DESCRIPTION

[0046] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0047] Example 1 Gene Design and Synthesis

[0048] (1) Genetic design:

[0049] According to the sequence characteristics of human type II collagen (UniProtKB / Swiss-Prot: P02458.3), the collagen sequence HC72 was designed, and its amino acid sequence is shown in SEQ ID NO.1. Its N-terminus starts with methionine to reduce the degradation rate of the protein; at the same time, a histidine tag is added to facilitate identification and purification. The selected sequence contains integrin binding sites: 2 RGD and 1 GAPGER. The amino acid sequence of the recombinant human type II collagen fragment is as follows:

[0050] SEQ ID NO.1

[0051] MHHHHHHGDRGDVGEKGPEGAPGKDGGRGLTGPIGPPGPAGANGEKGEVGPPGPAGSAGARGAPGERGETGPPGPAGFAGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGP KGARGAQGPPGATGFPGAAGRVGPPGSNGNPGPPGPPGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGLAGQRGIVGLPGQRGERGFPGLPGPSGEPGKQGAP

[0052] The coding nucleic acid sequence was reversely designed using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for host E. coli expression. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human type II collagen was obtained, and the nucleotide sequence corresponding to the above amino acid sequence is shown in SEQ ID NO.2:

[0053] ATGCACCACCATCACCACCATGGGGATAGGGGAGACGTTGGTGAGAAAGGTCCGGAAGGCGCTCCGGGCAAAGACGGCGGTCGTGGTTTGACCGGTCCGATTGGCCCGCCGGGCCCGGCGGGCGCGAACGGTGAGAAGGGCGAAGTTGGTCCGCCGGGTCCGGCGGGTTCTGCAGGTGCTCGCGGCGCGCCGGGCGAGCGCGGTGAAACCGGTCCGCCGGGCCCGGCGGGCTTCGCCGGCCCGCCGGGCGCTGATGGTCAGCCGGGCGCCAAAGGCGAGCAGGGTGAAGCAGGCCAAAAAGGTGATGCGGGTGCGCCAGGTCCGCAGGGCCCGTCCGGTGCGCCTGGCCCGCAAGGTCCAACGGGGGTGACTGGTCCGAAAGGTGCGCGTGGTGCGCAGGGTCCGCCGGGTGCTACCGGTTTTCCGGGCGCGGCAGGTAGAGTCGGTCCACCGGGCAGCAATGGAAACCCGGGCCCTCCGGGTCCGCCTGGCCCGTCGGGCAAGGACGGCCCCAAGGGTGCCCGTGGCGACAGCGGTCCACCGGGACGCGCAGGCGAACCGGGTCTGCAGGGCCCGGCGGGCCCGCCGGGCGAGAAGGGTGAGCCGGGTGATGATGGTCCGAGCGGTGCTGAAGGTCCGCCAGGCCCGCAAGGTCTGGCAGGTCAACGTGGGATCGTGGGCCTGCCGGGTCAACGTGGTGAACGTGGTTTCCCGGGCCTTCCGGGACCGAGCGGCGAGCCGGGTAAGCAGGGCGCGCCATAA

[0054] (2)Gene synthesis:

[0055] According to the above nucleic acid sequence, GenScript Biotech Corporation synthesized a nucleic acid fragment encoding a recombinant human type II collagen fragment.

[0056] Example 2 Construction of expression vector pET30a(+)-HC72

[0057] The nucleic acid fragment SEQ ID NO.2 obtained in Example 1 was ligated to the pET30a(+) plasmid (via the NdeI and XhoI multiple cloning sites) to obtain the recombinant plasmid pET30a(+)-HC72.

[0058] Example 3 Construction of expression strain BL21(DE3) / pET30a(+)-HC72

[0059] The construction of the expression strain refers to the method described in the "Molecular Cloning Laboratory Guide (3rd Edition)" (written by J. Sambrook et al.), and the specific steps are as follows: pick a single colony of Escherichia coli BL21 (DE3) and inoculate it into an LB test tube, then culture it at 37°C with shaking overnight; add 0.5ml of the overnight culture solution to a conical flask containing 50ml LB, and culture it at 37°C with vigorous shaking for about 2 hours to allow the bacteria to grow to the early logarithmic phase; transfer the bacteria to an ice-cold 50ml polypropylene tube under sterile conditions and place it on ice for 10 minutes; centrifuge at 4°C, 4000rpm, pour out the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible; add 6ml of ice-cold 0.1mol / L CaCl2 to resuspend the precipitate, and place it on ice for 30 minutes; centrifuge at 4°C, 3000rpm, pour out the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible; add 1.2ml of ice-cold 0.1mol / L Resuspend the precipitate with CaCl2 (if you want to prepare competent cells for storage at -70°C, add 0.1 mol / L CaCl2 containing 20% ​​glycerol to suspend the bacteria), place at 4°C for 5 to 24 hours, then take 200 μl of the competent cell suspension, add the recombinant plasmid pET30a(+)-HC72 prepared in Example 2 (volume <10 μl, DNA <50 ng), mix gently, and place on ice for 30 minutes; heat shock in a 42°C water bath for 90 seconds, and immediately cool on ice; add 500 μl of liquid LB culture medium, mix, and place in a 37°C shaker for low-speed shaking for recovery for 45 minutes (you can also add LB and directly place in a 37°C water bath for recovery for 1 hour, and shake the tube in the middle to suspend the cells); take the transformed cells and spread them on a plate with antibiotics (kanamycin), place them in a 37°C incubator and invert them for culture, and the colonies that grow are the expression strain BL21(DE3) / pET30a(+)-HC72.

[0060] Example 4 Induced expression of expression strain BL21(DE3) / pET30a(+)-HC72

[0061] A single colony of the expression strain BL21(DE3) / pET30a(+)-HC72 prepared in Example 3 was selected and placed in LB liquid medium containing 50 μg / mL Kan. After culturing overnight at 37°C and 200 r / min, it became an activated seed. Then, it was inoculated into a 5L fermenter containing 3L CM medium at a 3% inoculum. The fermentation process was controlled at 37°C, 30% dissolved oxygen, and pH 7.0. When OD 600 When the expression reached 60, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM to induce expression, and the culture was continued for 12 hours, and the cells were collected by centrifugation.

[0062] Example 5 Purification of recombinant human type II collagen HC72

[0063] The theoretical isoelectric point of HC72 is 6.31, and the pH of the buffer solution is 8.0. The specific purification steps are as follows:

[0064] 1. Breaking bacteria

[0065] The bacterial cells prepared in Example 4 were resuspended in an appropriate amount of lysis solution (20 mM Tris 500 mM NaCl, 20 mM imidazole, pH 8.0), with the ratio (m / v) of bacterial cells to lysis solution being 1:5-1:20, and 1:10 was selected in this example. The bacterial cells were ruptured using a high-pressure homogenizer. The ruptured bacterial solution was centrifuged at 12000 g for 1 h, and the supernatant was collected and filtered using a 0.45 μm filter membrane.

[0066] 2. Affinity chromatography

[0067] (1) Column balance:

[0068] The Ni Sepharose FF column was equilibrated with buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0) until the baseline was stable.

[0069] (2) Loading:

[0070] The supernatant was added to the chromatography column, and the column retention time was controlled to be no less than 5 min.

[0071] (3) Column flushing:

[0072] Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0073] (4) Target protein elution:

[0074] The solution was eluted with 80% Buffer A and 20% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type II collagen HC72.

[0075] 3. Ion exchange chromatography

[0076] (1) Sample processing:

[0077] The affinity chromatography eluate was diluted with ion exchange solution A (20 mM Tris, pH 8.0) or ultrafiltered to a conductivity of less than 5 mS / cm.

[0078] (2) Column balance:

[0079] The Q Sepharose FF column was equilibrated with buffer A until the baseline was stable.

[0080] (3) Loading:

[0081] The renaturation solution was added to the chromatography column, and the column retention time was controlled to be no less than 5 minutes.

[0082] (4) Column flushing:

[0083] Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0084] (5) Target protein elution:

[0085] The solution was eluted with 75% Buffer A and 25% Buffer B (20 mM Tris, 1 M NaCl, pH 8.0), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type II collagen HC72.

[0086] 4. Desalination

[0087] The HC72 solution was replaced with purified water by equal volume replacement using a 5 kDa ultrafiltration membrane device to remove salt from the solution, and then filtered with a 0.22 μm filter membrane and freeze-dried to obtain a recombinant collagen freeze-dried product.

[0088] Example 6 SDS-PAGE protein electrophoresis detection

[0089] Sample treatment: Collect the bacteria prepared in Example 4 and the purified samples in Example 5, add loading buffer and mix evenly, boil in a boiling water bath for 10 min, cool naturally and set aside. Select GenScript SurePAGE™ precast gel (4-12%) for sample loading, and perform electrophoresis at 140 V for 45-55 min until the bromophenol blue band runs to the bottom of the gel.

[0090] Coomassie Brilliant Blue R-250 staining using a microwave oven: (1) Prepare the staining solution: Dissolve Coomassie Brilliant Blue R-250 to a final concentration of 0.1% (W / V) in 40% ethanol and 10% acetic acid solution; (2) Prepare the destaining solution: Dissolve 10% (V / V) ethanol and 7.5% (V / V) acetic acid together; (3) After electrophoresis is completed, pry open the gel sheet, remove the gel, and then place it in a staining container containing 100 ml of staining solution; (4) Cover the container with a lid and place it in a microwave oven and heat it on high for 8 minutes. To avoid danger, be careful not to let the solution boil; (5) Remove the staining container from the microwave and place it on a decolorizing shaker at room temperature for 5 minutes; (6) Pour out the staining solution and carefully wash the gel with deionized water; (7) Pour out the deionized water and add 100 ml of decolorizing solution; (8) Cover the container with a lid and place it in a microwave oven on high heat for 8 minutes; (9) Pour out the decolorizing solution, add new decolorizing solution, and repeat step 8; (10) Remove from the microwave and place it on a decolorizing shaker at room temperature and gently shake until the background is clear.

[0091] The results are shown in the attached figure Figure 1-3 shown.

[0092] Example 7 Extraction of bovine cartilage collagen

[0093] For the beef rib cartilage, remove the periosteum on the surface of the cartilage, clean it, cut it into small pieces, and defat it with chloroform and methanol. Then decalcify it with 0.1 M hydrochloric acid, and then add 4M NaCl-20mM Tris-HCl buffer to remove soluble proteins; then use 4M guanidine hydrochloride-20mM Tris-HCl solution to remove insoluble proteins; then use 3% pepsin to enzymatically hydrolyze overnight. After the above solution is centrifuged at 12000g for 5 minutes, take the supernatant for dialysis or ultrafiltration desalination and remove small molecular impurities to obtain type II collagen.

[0094] Effect Example 1 Cytotoxicity Experiment of Recombinant Human Type II Collagen HC72

[0095] The cytotoxicity of HC72 was evaluated according to the method specified in the MTT cytotoxicity test in Appendix C of GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test. The experiment was divided into four groups: sample group (recombinant human type II collagen HC72 solution prepared in Example 5), positive control group (extract of ZDEC Polyurethane Film), negative control group (extract of High Density Polyethylene Film), and medium control group (cell culture medium containing 10% serum, placed at 37±1°C, 24±2h). L929 cells were cultured in the extract or cell culture medium described in each group, and the relative cell proliferation rate was calculated when the negative control group and the positive control group met the requirements. Figure 4 As shown, HC72 had no obvious cytotoxicity in the concentration range of 0.1 mg / ml~10 mg / ml.

[0096] Effect Example 2 Recombinant human type II collagen HC72 promotes cell proliferation test

[0097] Bone marrow mesenchymal stem cells were collected at 5×10 3 / well for cell inoculation. After the cells were inoculated in the 96-well culture plate, it was placed in a 37°C, 5% CO2 incubator and cultured for 24 hours. The culture fluid in the wells was discarded, 100 μL of culture medium was added to each well of the blank control group, 100 μL of HC72 solution was added to each well of the sample group, and 100 μl of bovine type II collagen obtained in Example 7 was added to the positive control group. The 96-well culture plate was placed in a 37°C, 5% CO2 incubator and cultured for 24 hours. 10 μL of CCK8 reagent was added to each well and placed in the incubator for incubation for 2 hours. After incubation, the absorbance value of each well at a wavelength of 450 nm was detected using an enzyme marker. Figure 5 As shown, recombinant human type II collagen HC72 has a good cell proliferation effect, and has no significant difference in promoting cell proliferation compared with bovine type II collagen.

[0098] Effect Example 3 Study on the stability of recombinant human type II collagen

[0099] The recombinant human type II collagen freeze-dried product was placed at room temperature to investigate the stability of the product. Samples were taken at different time points of 0 month, 1 month, 3 months, 6 months, and 12 months for SDS-PAGE gel electrophoresis. Figure 6 As shown, there is no obvious change in the target band, indicating that the product has good stability and is suitable for industrial production and application.

[0100] The above embodiment provides a recombinant human type II collagen that can be produced in a technological manner. The recombinant human type II collagen is non-cytotoxic and can promote the proliferation of bone marrow mesenchymal stem cells, and is an ideal raw material for bone tissue repair products.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human type II collagen, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. A recombinant human type II collagen according to claim 1, characterized in that: The nucleotide sequence of the recombinant human type II collagen is shown in SEQ ID No.

2.

3. A carrier, characterized in that The vector contains the nucleotide sequence shown in SEQ ID No.

2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3.

5. A method for preparing the recombinant human type II collagen as claimed in claim 1, characterized in that: The specific steps include: (1) Protein sequence design Based on human type II collagen, an amino acid sequence as shown in SEQ No. 1 was designed; (2) Gene design and synthesis Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human type II collagen described in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type II collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type II collagen fragment; (3) Construction of expression vector: The expression plasmid was obtained by ligating the plasmid to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites; (4) Construction and screening of expression strains: The plasmid described in step (3) is transferred into the host cell competent state by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Inducing the expression strain obtained in step (4) to express, and collecting the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption and centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a recombinant human type II collagen freeze-dried product.

6. The preparation method according to claim 5, characterized in that: The host cell is Escherichia coli BL21 (DE3).

7. Use of the recombinant human type II collagen as claimed in claim 1 in preparing a bone tissue repair product.

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