Recombinant human-derived II-type collagen, nucleic acid molecule, carrier, cell, composition and application of recombinant human-derived II-type collagen, nucleic acid molecule, carrier, cell and composition

Abstract

The invention discloses a recombinant human-derived II-type collagen, a nucleic acid molecule, a carrier, a cell, a composition and application thereof, and relates to the technical fields of genetic engineering and synthetic biology. The recombinant human-derived II-type collagen comprises a calcium ion binding domain and has an effect of promoting osteoblast proliferation, so that the recombinant human-derived II-type collagen has a good application prospect in preparation of medical instruments and compositions. The recombinant human II-type collagen provided by the invention can be expressed in a common expression system, and has the advantages of high expression quantity and no endotoxin hidden danger. Moreover, the recombinant human II-type collagen does not carry purification labels such as histidine labels and the like, and can be purified through a molecular sieve, so that the target protein can be directly obtained finally, and the purification labels such as the histidine labels and the like do not need to be additionally cut off. The production method of the recombinant human-derived II-type collagen is suitable for industrial large-scale production, and the prepared product does not have an animal-derived infection source, so that the biological safety is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and synthetic biology, and in particular to a recombinant human type II collagen, a nucleic acid molecule, a vector, a cell, a composition and applications thereof. Background Art

[0002] Collagen is a type of protein widely distributed in human connective tissue. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. At present, about 20 types of collagen have been discovered. Collagen is the scaffold of structures such as basement membranes, bones, and cartilage, and plays a role in supporting, protecting, binding, and forming barriers for the body and organs. In addition to its biomechanical role, it can also transmit signals between cells through extracellular matrix receptor molecules and participate in regulating cell behavior. Collagen can directly bind to cell surface receptors, or indirectly bind to other intermediary molecules to participate in cell surface receptor recognition and signal transduction processes. Collagen also plays a very important role in its dependent dynamic cell activities, such as tissue reconstruction, including morphological processes and wound healing. It also plays an important role in immune system regulation, hemostasis, and thrombosis.

[0003] Recombinant human collagen produced by genetic engineering is a technology that transfers human collagen genes into engineered cells after specific sequence design, enzyme cleavage and splicing, and connection to vectors, and then produces collagen and its analogs through fermentation expression. At present, there are many methods for producing recombinant human collagen through genetic engineering, and each method comprehensively involves different expression systems such as Escherichia coli, yeast, insect cells, and transgenic crops. Due to the high cost and long cycle of animal cell expression systems, and the difficulty, high cost, and extremely low yield of collagen extraction and purification technology of transgenic crops, the recombinant collagen that can enter industrial production or commercialization mainly adopts technology based on microbial fermentation such as Escherichia coli and yeast, and the specific products include full-length / non-full-length recombinant human collagen of type I or type III. Compared with natural collagen from animal tissue, recombinant collagen fermented by microorganisms has better processability (such as water solubility and emulsification properties), can better control the risk of pathogenic virus transmission, and can reduce immunogenicity as much as possible through sequence design.

[0004] Type II collagen is the main collagen component of cartilage matrix and plays an important role in maintaining the compactness of cartilage structure and biomechanical properties.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a recombinant human type II collagen, a nucleic acid molecule, a vector, a cell, a composition and its application to solve the above-mentioned technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a recombinant human type II collagen, the amino acid sequence of which is a segment extracted from the full-length sequence, and the amino acid sequence of which is located at the 579th to 1265th amino acid sequence of natural human type II collagen.

[0009] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned recombinant human type II collagen.

[0010] In a third aspect, the present invention provides a vector comprising the isolated nucleic acid molecule described above.

[0011] In a fourth aspect, the present invention further provides a host cell, comprising: the above-mentioned vector.

[0012] In a fifth aspect, the present invention also provides the use of recombinant human type II collagen in the preparation of medical devices and compositions.

[0013] In a sixth aspect, the present invention also provides a composition comprising the above-mentioned recombinant human type II collagen.

[0014] In a seventh aspect, the present invention also provides a method for producing the above-mentioned recombinant human type II collagen, which comprises: culturing the above-mentioned host cells.

[0015] In an eighth aspect, the present invention further provides the use of recombinant human type II collagen, isolated nucleic acid molecules, vectors, host cells, and / or the above-mentioned compositions in preparing a kit, wherein the kit is used for at least one of the following purposes:

[0016] (1) Increase cell adhesion; (2) Cartilage repair; (3) Increase bone adhesion; (4) Prevent and treat osteoporosis; (5) Promote osteoblast proliferation.

[0017] The present invention has the following beneficial effects:

[0018] (1) The recombinant human type II collagen provided by the present invention includes the calcium ion binding domain of natural human type II collagen, does not contain the amino acid sequences at the C-terminus and N-terminus of the collagen region, is a truncated protein of natural human type II collagen, and has the advantage of low immunogenicity.

[0019] (2) The calcium ion binding domain in recombinant human type II collagen has the effect of promoting osteoblast proliferation. Therefore, recombinant human type II collagen has good application prospects in the preparation of medical devices and compositions.

[0020] (3) The recombinant human type II collagen provided by the present invention can be expressed in a common expression system, especially in Pichia pastoris, and has the advantages of high expression level and no endotoxin risk. Moreover, the recombinant human type II collagen may not carry purification tags such as histidine tags, and can be purified by molecular sieves to obtain the target protein directly without the need for additional removal of purification tags such as histidine tags.

[0021] (4) The method for producing recombinant human type II collagen of the present invention is suitable for large-scale industrial production, and the prepared product has no animal-derived infection source, and therefore has higher biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is the SDS-PAGE electrophoresis diagram of the supernatant of cell disruption expressing recombinant human type II collagen in Example 1;

[0024] Figure 2 This is the SDS-PAGE electrophoresis diagram of the recombinant human type II collagen purified in Example 2;

[0025] Figure 3 These are microscopic images of cell adhesion test (A: before centrifugation; B: after centrifugation - recombinant type II collagen; C: after centrifugation - PBS). DETAILED DESCRIPTION

[0026] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0027] In a first aspect, the present invention provides a recombinant human type II collagen, whose amino acid sequence includes: the amino acid sequence from positions 579 to 1265 of natural human type II collagen, and the amino acid sequence of natural human type II collagen is shown in SEQ ID NO.3.

[0028] After screening, the inventors found that after deleting the amino acids at the N-terminus of positions 1 to 578 and the amino acids at the C-terminus of position 1266 of natural human type II collagen, the remaining amino acids still have functional active regions such as calcium ion binding domains and have a triple helix structure. Among them, the D, N, Q, C, and D sites at the position of DPNQGCTLD in the sequence are all calcium ion binding sites. The calcium ion binding domains formed by these calcium ion binding sites have the effect of promoting osteoblast proliferation, can increase the number of bone cells generated, and keep bone growth in a benign state. Therefore, recombinant human type II collagen has good application prospects in the preparation of medical devices and compositions. In addition, recombinant human type II collagen can effectively strengthen bone and joint function, increase bone adhesion, prevent osteoporosis, and improve human immunity.

[0029] The recombinant human type II collagen provided by the present invention is a truncated protein of natural human type II collagen and has the advantage of low immunogenicity.

[0030] In a preferred embodiment of the present invention, the amino acid sequence of the recombinant human type II collagen is shown as SEQ ID NO.1.

[0031] The specific amino acid sequence information of SEQ ID NO.1 is as follows:

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。

[0033] The amino acid sequence of natural human type II collagen is shown in SEQ ID NO.3:

[0034] MFRFVDARTFVLLAATQAILLTVVKCQHSHTQNLKSTCIQDGQTYSDKDVWKPRPCSICVCDTGIILCDDIICEDIRDCPNPEIPFGECCPVCLNQRPPPIDRQRGPRGQKGEPGDIKDVVGPNGPPGPQGPAGEQGVRGL RGEKGEMGAAGPRGRDGEPGTPGNPGPPGLPGTSGRPDFGGSFASQMARGFDEKAGGAQMGVMQGPMGPMGPRGPPGPTGVPGPQGFQGSAGEAGESGAAGPMGPRGPPGPSGKPGDDGEAGKAGKPGDRGPPGPQGARGFP GTPGLPGVKGHRGYSGLDGAKGETGAAGAKGESGAAGENGAPGPMGPRGLPGERGRPGSPGSAGARGNDGLPGPAGPPGPVGPAGAPGFPGAPGSKGEAGPTGARGPEGAQGSRGESGTPGSPGPAGAPGNPGTDGIPGSKGSAGAPGIAGAPGFPGPRGPPGPQGATGPLGPKGTQGDPGATGFKGETGPKGEIGPVGPQGAPGPAGEEGKRGPRGEPGAAGPNGPPGERGAPGNRGFPGQDGLAGPKGAPGERGVAGVSGPKGANGDPGRPGEPGLPGARGL.

[0035] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned recombinant human type II collagen.

[0036] The term "nucleic acid molecule encoding recombinant human type II collagen" may include a polynucleotide encoding the recombinant human type II collagen of the present invention, or may include additional coding and / or non-coding sequences.

[0037] When the present invention provides the above amino acid sequence, those skilled in the art can easily obtain the nucleic acid sequence encoding the above recombinant human type II collagen based on the degeneracy of codons.

[0038] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0039] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0040] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0041] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0042] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0043] In an alternative embodiment, the nucleic acid molecule is selected from DNA, RNA, or a combination thereof.

[0044] The nucleic acid molecule may or may not contain nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, or nucleotides encoding a leader sequence to facilitate purification or secretion of the polypeptide.

[0045] In a third aspect, the present invention provides a vector comprising the isolated nucleic acid molecule described above.

[0046] Vectors include but are not limited to expression vectors, shuttle vectors, and integration vectors.

[0047] The term "expression vector" refers to bacterial plasmids, yeast plasmids or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0048] In a preferred embodiment of the present invention, the vector is a bacterial vector or a fungal vector.

[0049] Transformation of host cells with vectors can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0050] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0051] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0052] In an optional embodiment, the expression vector further comprises a signal peptide, such as a nuclear localization signal peptide.

[0053] In a preferred embodiment of the present invention, the vector is an expression vector.

[0054] In a preferred embodiment of the present invention, the vector comprises an expression control element operably linked to the isolated nucleic acid molecule;

[0055] In a preferred embodiment of the present invention, the expression control element is a promoter, a terminator and / or an enhancer.

[0056] In a fourth aspect, the present invention further provides a host cell, comprising: the above-mentioned vector;

[0057] In a preferred embodiment of the present invention, the host cell is selected from bacteria, fungi, mammalian cells or insect cells;

[0058] In a preferred embodiment of the present invention, the bacterium is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis; in a specific embodiment, the host cell is Escherichia coli BL21.

[0059] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0060] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parental cells in nucleic acid content, but may contain mutations.

[0061] Trichoderma reesei is a food-grade safety strain certified by the US FDA. As a production strain, this strain does not produce endotoxins similar to those produced by Escherichia coli. Compared with brewer's yeast fermentation, the fermentation process of Trichoderma reesei does not produce a large amount of heat, reducing the investment cost of cooling equipment. Compared with large fungi, the fermentation cycle of Trichoderma reesei is shorter.

[0062] In a preferred embodiment of the present invention, the yeast is selected from at least one of the following genera: Dekkera, Brettanomyces, Hansenula, Kluyveromyces, Pichia, Candida, Kluyveromyces, Debaryomyces, Kazachstania, Wickerhamomyces, Lindnera, Zygotorulaspora, Zygosaccharomyces and Schizosaccharomyces.

[0063] In a preferred embodiment of the present invention, the insect cells are insect ovary cells.

[0064] The above-mentioned recombinant bacteria (or genetically engineered bacteria) are obtained by transforming a recombinant expression vector into a host microorganism by conventional methods in the art; the host microorganism can be various host microorganisms conventional in the art, as long as the recombinant expression vector can stably replicate itself and the gene encoding the recombinant human type II collagen carried by it can be effectively expressed.

[0065] In a preferred embodiment of the present invention, the recombinant bacteria refers to at least one of resting cells of the recombinant bacteria, living cells of the recombinant bacteria, dead cells of the recombinant bacteria, and cell fragments of the recombinant bacteria.

[0066] Dormant cells, also known as resting cells, are a special cell state. In this state, cells do not grow and reproduce, but still contain various enzyme systems and have oxidation and fermentation capabilities. Under suitable conditions, dormant cells can resume growth. The characteristics of dormant cells include: a. Cells maintain growth potential: Although in a dormant state, these cells can re-enter the cell cycle and restore their proliferation ability when given appropriate stimulation. b. Strong specificity: Dormant cells have strong specificity in the reaction and can improve the substrate conversion rate. c. Not easy to be contaminated by foreign bacteria: Due to its characteristics, dormant cells can reduce the inhibition of bacterial growth and enzyme synthesis by products during use.

[0067] Dead bacteria of recombinant bacteria include but are not limited to bacteria obtained by inactivation by heat, pressure, radiation, etc.

[0068] Cell disruption products refer to: including but not limited to, cell disruption products obtained by changing the permeability of cell membranes through ultrasonic, mechanical, chemical, biological and other means, resulting in leakage of cell contents.

[0069] In a preferred embodiment of the present invention, the dead bacteria are selected from at least one of a precipitate of dead bacteria and a cell-free supernatant of dead bacteria. The cell-free supernatant of dead bacteria refers to the "exudate contents" remaining after removing the outer shell of the dead bacteria.

[0070] In a fifth aspect, the present invention also provides the use of recombinant human type II collagen in the preparation of medical devices and compositions;

[0071] In a preferred embodiment of the present invention, the medical device is selected from at least one of artificial bone, artificial cornea and vitreous body;

[0072] In a preferred embodiment of the present invention, the artificial bone is artificial cartilage;

[0073] In a preferred embodiment of the present invention, the composition is used for at least one of the following purposes:

[0074] (1) Increase cell adhesion; (2) Cartilage repair; (3) Increase bone adhesion; (4) Prevent and treat osteoporosis; (5) Promote osteoblast proliferation.

[0075] In a preferred embodiment of the present invention, the composition is a pharmaceutical composition or a health product.

[0076] In a sixth aspect, the present invention also provides a composition comprising the above-mentioned recombinant human type II collagen.

[0077] The composition can be a pharmaceutical composition, a human structural material, etc., and can function as a tissue filler, a cartilage repair agent, etc. The composition of the present invention can be injectable.

[0078] In a seventh aspect, the present invention also provides a method for producing the above-mentioned recombinant human type II collagen, which comprises: culturing the above-mentioned host cells.

[0079] In an optional embodiment, the host cells and / or culture medium containing the recombinant human type II collagen are harvested; and the recombinant human type II collagen is purified.

[0080] In a specific embodiment, the host cell is Escherichia coli. After obtaining the genetically engineered Escherichia coli, the specific culture conditions are as follows: the genetically engineered bacteria are inoculated in a seed culture medium, and after culturing for 10 to 12 hours at 34°C, as the seed solution of the upper tank, the seed solution is expanded and cultured at 10% inoculum to be accessed into an NBS 415 fermenter with an initial volume of 5L, and cultured at 34°C, the ventilation flow rate is 1vvm without load, and the batch stage is ensured by increasing the speed to ensure that the dissolved oxygen (DO) in the fermenter is above 25% air saturation, and the pH of the fermentation liquid is controlled to be 6.5 by 3mol / L NaOH feedback in the batch stage, and the pH of the fermentation liquid is controlled to be 6.8 by 25% ammonia (w / w) in the feed stage. After the batch glucose is exhausted, glucose is added by pulse addition. When OD600 reaches about 100, it is heated to 42°C to induce expression of type II collagen.

[0081] The present invention does not impose any limitation on the separation and purification method, which can be determined according to the method, for example, salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method can be used.

[0082] In an eighth aspect, the present invention further provides the use of recombinant human type II collagen, isolated nucleic acid molecules, vectors, host cells, and / or the above-mentioned compositions in preparing a kit, wherein the kit is used for at least one of the following purposes:

[0083] (1) Increase cell adhesion; (2) Cartilage repair; (3) Increase bone adhesion; (4) Prevent and treat osteoporosis; (5) Promote osteoblast proliferation.

[0084] The kit can be a kit for tissue filling and / or volume expansion, or a kit for detecting recombinant human type II collagen, such as setting an isolated nucleic acid molecule as a positive control.

[0085] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0086] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0087] Example 1

[0088] This example provides an expression vector for recombinant human type II collagen, and the construction method thereof is as follows:

[0089] The nucleotide sequence SEQID NO.2 of recombinant type II collagen was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. The restriction sites Nco I and Xho I were added to both ends of the nucleotide sequence and directly connected to the pET32a vector by Sangon Biotech (Shanghai) Co., Ltd. It was named pET32a-Ⅱ.

[0090] The specific nucleotide sequence information of SEQ ID NO.2 is as follows:

[0091]

[0092] Example 2

[0093] This embodiment provides a method for constructing and expressing (or producing) recombinant type II collagen engineering bacteria.

[0094] Escherichia coli BL21 was used as the expression host bacteria, and the pET32a-Ⅱ plasmid obtained in Example 1 was transformed into Escherichia coli BL21 by electroporation. After the construction of the plasmid transformation strain was completed, the successfully transformed strains were screened by the expression of the exogenous marker gene and 5 successfully transformed single colonies were picked. Then, the 5 single colonies picked from each transformed strain were cultured in a shake flask to detect the expression of the target protein. After comparison, a strain with the highest type II collagen production was screened and named E. coli BL 21HLCII.

[0095] The genetically engineered bacteria obtained above were streaked and transferred to the plate, placed in a biochemical incubator at 34°C for 24 hours, and then a single colony of appropriate size was selected for secondary streaking and transfer. After culturing at 34°C for 12 hours, the plate was taken out and sealed and stored in a refrigerator at 4°C. The seed plate was taken out from the refrigerator at 4°C, and 1-2 single colonies were selected and transferred to a shake bottle containing 80mL of seed culture solution. After culturing at 34°C for 10-12 hours, the plate was transferred to another 8 shake bottles containing 80mL of seed solution, and cultured at the same temperature for 10-12 hours to prepare the jar fermentation seed solution.

[0096] Inoculate 800 mL of the cultured seed liquid into a fermenter containing 14 L of fermentation medium and culture at 34°C with an air flow rate of 1 vvm at no load. In the batch stage, increase the rotation speed to ensure that the dissolved oxygen (DO) in the fermenter is above 25% air saturation. Since organic acids will be produced during the entire fermentation process, 3 mol / L NaOH is used to feedback control the pH of the fermentation liquid to 6.5 in the batch stage. 25% ammonia water (w / w) is used to control the pH of the fermentation liquid to 6.8 in the feeding stage. After the glucose in the batch is consumed, pulse addition of glucose is started. The carbon-nitrogen ratio added in the feeding stage is guaranteed to be the same under different pulse intensities. When OD 600 When the temperature reaches about 100, the temperature is raised to 42°C to induce the expression of type II collagen. When it is found that the cells stop growing as the induction proceeds, that is, the OD begins to decrease, indicating that the fermentation is over, the bacteria are collected by centrifugation.

[0097] Example 3

[0098] This example provides a method for purifying recombinant type II collagen.

[0099] 1. Bacterial cell disruption

[0100] The cell disruption process adopts high pressure homogenization batch cycle, homogenization twice, the disruption power is selected to be 70MPa, and the concentration of the bacterial suspension is selected to be: the mass of the bacteria collected in Example 2 and the volume ratio of water is 1:10. The cell disruption supernatant is collected by centrifugation and detected by SDS-PAGE electrophoresis. The molecular weight of type II collagen is about 75kDa. Figure 1 As shown, the supernatant in the figure is two parallel samples.

[0101] 2. NaCl salting out precipitation

[0102] Add 5% NaCl to the supernatant collected in the previous step, maintain the temperature at 18°C, adjust the pH to 2.8 with 6 mol / L HCl, and perform salting precipitation under this condition. The supernatant after precipitation is ultrafiltered, concentrated and desalted using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa.

[0103] 3. Freeze Drying

[0104] The protein was concentrated to 20%-30% of the initial volume by ultrafiltration, and then pre-frozen in a -20°C refrigerator for 4 hours. It was then transferred to a vacuum freeze dryer for freeze-drying. The freeze-dried protein was collected after 48 hours and stored in a -80°C refrigerator for later use.

[0105] Figure 2 The figure is the SDS-PAGE electrophoresis of the protein sample. After SDS-PAGE electrophoresis detection and Coomassie Brilliant Blue staining, a single band was obtained, and it can be seen that the molecular weight of recombinant type II collagen is about 75 kDa, which is consistent with the protein sequence deduction.

[0106] Example 4

[0107] In this example, the recombinant type II collagen provided in Example 3 was used to conduct an experiment to promote chondrocyte proliferation.

[0108] Experimental groups: The experimental settings were recombinant type II collagen (group 1), full-length type II collagen α1 chain collagen (group 2), and a blank group (α-MEM culture medium).

[0109] 1) Group 1 and Group 2 were added with α-MEM medium for extraction for 24 hours. The extract was filtered with a sterile filter and diluted to 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL for later use;

[0110] 2) The third generation of rat articular chondrocytes were cultured until the cell bottle was full, digested with trypsin, and counted. 4100 μL of cells were inoculated into a 96-well plate at a cell concentration of 100 μL / mL. After culturing for 24 hours, the culture medium was aspirated and the samples of group 1 and group 2 prepared in step 1) were added to each well (the concentrations of each sample were set to 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL, respectively). A total of 12 samples were added, each with 6 replicates. The culture was continued for 24 hours, 48 ​​hours, and 72 hours, and the extraction growth medium was replaced every day. The cell proliferation was detected by MTT method at 24 hours, 48 ​​hours, and 72 hours of culture.

[0111] Cell viability = Experimental group A 570 ×100% / blank group.

[0112] Table 1 Proliferation-promoting effects of different collagen samples at different concentrations on rat articular chondrocytes

[0113]

[0114] As shown in Table 1, recombinant type II collagen (Group 1) and full-length type II collagen α1 chain collagen (Group 2) both have a proliferative effect on rat articular chondrocytes, among which the cell proliferation rate of recombinant type II collagen was significantly higher than that of full-length type II collagen α1 chain collagen (P < 0.05).

[0115] Example 5

[0116] In this example, the recombinant type II collagen provided in Example 3 was used to perform an experiment on promoting chondrocyte cell adhesion.

[0117] The experimental method is as follows:

[0118] 1. Coating preparation: Add 100 μL of standard solution, collagen sample, and PBS blank control to a 96-well plate (the test samples in this test are PBS, standard type I collagen sample (manufacturer Sigma, product number C9879), and recombinant type II collagen; the concentration of each sample is 1 mg / mL). Prepare 4 wells for each sample coating and incubate in a 37°C, 5% CO2 incubator for 1 hour. Remove excess coating solution from the wells, add 100 μL of 1% BSA-PBS solution, and incubate in a 37°C, 5% CO2 incubator for 1 hour. After removing the liquid in the wells, wash three times with D-PBS, discard the cleaning solution, seal with sealing film, and place at 4°C for use.

[0119] 2. Cell preparation: The third generation rat articular chondrocytes were cultured in a 37°C, 5% CO2 cell culture incubator, and the cell density and state were observed under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. The cells were diluted to 1×10 using complete culture medium premixed with Hoechst 33342 fluorescent dye (10%). 4100 μL of cells were added to the wells, covered with aluminum foil, and incubated for 1 h at 37°C, 5% CO2. Three replicates were measured, and the fourth well was used to adjust the microscope parameters and its measurement value was not used.

[0120] 3. Detection: Use an inverted microscope to capture at least 5×5 digital tile images (fluorescence) of each of the 3 wells. Each well is filled with D-PBS to form a "reverse meniscus", purged of bubbles and covered with acetate sealing tape. Centrifuge the plate (inverted) at 22°C for 5 min at an optimal relative centrifugal force (RCF) of 350g. After centrifugation, discard the acetate sealing tape and remove the supernatant from the well. After washing once with D-PBS, add 100 μL D-PBS. For each of the 3 wells, take a total of 25 fluorescent tile digital images (at least 5×5 matrix is ​​recommended, with 10% tile overlap). Count approximately 2400 to 3600 cells for each sample (800-1200 cells / well × 3 wells).

[0121] 3. Calculation of results

[0122] Determine the number of cells before and after centrifugation

[0123] The tiled images of each well are "stitched" together. Subsequently, an automated cell counting program (such as AxioVision) is used to count the number of fluorescently labeled cell nuclei. The percentage of adhesion is calculated according to formula 1:

[0124]

[0125] Where:

[0126] V——adhesion percentage;

[0127] Nt—cell number after centrifugation;

[0128] Nc - number of cells before centrifugation.

[0129] IV. Experimental Results

[0130] The results of this experiment refer to Figure 3 As shown:

[0131] Statistical results of cell adhesion count and adhesion rate (%):

[0132]

[0133] Conclusion: According to this experimental method, the cell adhesion of the tested samples, PBS blank control, and standard type I collagen were all 0, while the cell adhesion rate of recombinant type II collagen was 55.36%.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant human type II collagen, It is characterized in that The amino acid sequence of the recombinant human type II collagen is a segment of the full-length sequence, and the amino acid sequence is located at the 579th to 1265th amino acid sequence of the natural human type II collagen; Preferably, the amino acid sequence of the natural human type II collagen is as shown in SEQ ID NO.

3.

2. The recombinant human type II collagen according to claim 1, It is characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

3. An isolated nucleic acid molecule, It is characterized in that It encodes the recombinant human type II collagen described in any one of claims 1-2.

4. A carrier, It is characterized in that It comprises the isolated nucleic acid molecule of claim 3; Preferably, the vector is an expression vector; Preferably, the vector comprises an expression control element operably linked to the isolated nucleic acid molecule; Preferably, the expression control element is a promoter, a terminator and / or an enhancer.

5. A host cell, It is characterized in that It comprises: the vector according to claim 4; Preferably, the host cell is selected from bacteria, fungi, mammalian cells or insect cells; Preferably, the bacterium is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis; Preferably, the fungus is Trichoderma reesei or yeast.

6. Use of the recombinant human type II collagen according to any one of claims 1 to 2 in the preparation of medical devices and compositions; Preferably, the medical device is selected from at least one of artificial bone, artificial cornea and vitreous body; Preferably, the artificial bone is artificial cartilage; Preferably, the composition is used for at least one of the following purposes: (1) Increase cell adhesion; (2) Cartilage repair; (3) Increase bone adhesion; (4) Prevent and treat osteoporosis; (5) Promote osteoblast proliferation.

7. The use according to claim 6, It is characterized in that The composition is a pharmaceutical composition or a health product.

8. A composition, It is characterized in that It comprises the recombinant human type II collagen as described in any one of claims 1-2.

9. A method for producing the recombinant human type II collagen according to any one of claims 1 to 2, It is characterized in that It includes: Cultivate the host cell according to claim 5.

10. Use of the recombinant human type II collagen according to any one of claims 1 to 2, the isolated nucleic acid molecule according to claim 3, the vector according to claim 4, the host cell according to claim 5, and / or the composition according to claim 8 in preparing a kit, It is characterized in that The kit is used for at least one of the following purposes: (1) Increase cell adhesion; (2) Cartilage repair; (3) Increase bone adhesion; (4) Prevent and treat osteoporosis; (5) Promote osteoblast proliferation.

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