Recombinant Type II Humanized Collagen and Its Uses

Abstract

This disclosure provides recombinant type II humanized collagen and its uses. The recombinant type II humanized collagen has a typical triple helix structure and excellent biological properties, can be used for cartilage repair, and does not produce immune rejection or allergic reactions when applied to the human body.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to recombinant type II humanized collagen and its uses. Background Technology

[0002] Collagen is a type of protein that is widely distributed in human connective tissues and is the most abundant protein in the human body, accounting for 25% to 35% of the total protein. At least 28 collagen subtypes have been found in the human body, located in different tissues and organs.

[0003] Type II collagen is mainly found in cartilage tissue, vitreous humor, and cornea. It is a high-molecular-weight protein whose filamentous collagen fibers intertwine with elastin and polysaccharide proteins to form a network structure, also known as complex bone collagen. Type II collagen is an essential component for cartilage and bone formation, bone growth, and the maintenance of mature cartilage. Therefore, non-denatured collagen can serve as a structural and functional component of cartilage.

[0004] Type II collagen, a major component of articular cartilage matrix, works with lubricating components such as hyaluronic acid and proteoglycans to protect cartilage from wear and tear. With age, the rate of collagen synthesis gradually decreases, collagen loss intensifies, articular cartilage degenerates, and bone-on-bone friction increases, leading to joint inflammation. Currently, a common treatment for osteoarthritis is consuming collagen supplements; however, the low protein utilization rate results in slow cartilage regeneration, hindering patient recovery and affecting the treatment process and efficiency. Due to the unsatisfactory efficacy of current treatments, patients often ultimately have to opt for total joint replacement surgery, which is not only costly but also carries the risk of catastrophic complications such as postoperative thrombosis, postoperative infection, and even amputation.

[0005] Traditional methods for producing type II collagen involve treating animal-derived tissues with acids, alkalis, and enzymes to extract collagen derivatives. However, the collagen extracted using these methods loses its original biological activity and cannot function properly in the biomedical field. With the development of modern technology, some extraction methods have emerged both domestically and internationally for removing impurities from animal cartilage and extracting non-denatured type II collagen through enzymatic hydrolysis. However, these methods are time-consuming, result in low product purity, make it difficult to maintain the triple helix structure of collagen, and lead to poor product stability, hindering large-scale production. Furthermore, the immunogenicity of animal-derived collagen cannot be eliminated, which is a significant reason for its limited application. To address the shortcomings of existing technologies, some companies have proposed using Pichia pastoris as a host strain to prepare type II collagen for cartilage repair. However, this collagen is not humanized and therefore exhibits immunogenicity.

[0006] Therefore, there is an urgent need for a recombinant type II humanized collagen that can be directly injected into the human body without causing an immunogenic reaction, so as to be used as a human structural material for cartilage repair. Summary of the Invention

[0007] This invention utilizes synthetic biology and structural biology techniques to develop recombinant type II humanized collagen with a triple helix structure that can perform the functions of human collagen.

[0008] The first aspect of the present invention provides recombinant type II humanized collagen, comprising n repeating units connected directly or via a linker, the repeating units comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or a variant thereof, the variant being (1) an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO:1 or SEQ ID NO:3; or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1 or SEQ ID NO:3.

[0009] In some implementations, n is an integer from 1 to 20. In some implementations, n is 1, 4, 8, or 12.

[0010] In some embodiments, the linker comprises one or more amino acid residues; preferably, the linker comprises 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0011] In some embodiments, the collagen comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5.

[0012] In some embodiments, the collagen comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:4 or SEQ ID NO:5.

[0013] In some embodiments, the collagen comprises an amino acid sequence obtained by substitution, deletion, or insertion of one or more amino acids based on SEQ ID NO:4 or SEQ ID NO:5.

[0014] A second aspect of the present invention provides a fusion protein comprising the recombinant type II humanized collagen described in the first aspect of the present invention and a purified tag and / or precursor.

[0015] In some embodiments, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag, or NusA tag.

[0016] In some embodiments, the leader is selected from synthetic leader sequences, signal peptides, or leader peptides.

[0017] A third aspect of the invention provides a polynucleotide encoding the collagen described in the first aspect of the invention or the fusion protein described in the second aspect of the invention.

[0018] In some embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:8, or a degenerate sequence thereof.

[0019] A fourth aspect of the present invention provides a carrier comprising the polynucleotides described in the third aspect of the present invention.

[0020] In some embodiments, the vector is an expression vector; in some embodiments, the vector includes a control element operatively linked to a polynucleotide; in some embodiments, the control element is selected from promoters, terminators, and / or enhancers.

[0021] The fifth aspect of the present invention provides a host cell comprising the polynucleotides described in the third aspect of the present invention, or the vectors described in the fourth aspect of the present invention.

[0022] In some embodiments, the host cell is a bacterium, fungus, or animal cell; in some embodiments, the bacteria include Escherichia coli; in some embodiments, the fungus includes yeast, such as Saccharomyces cerevisiae.

[0023] The sixth aspect of the present invention provides a trimer collagen comprising the recombinant type II humanized collagen described in the first aspect of the present invention.

[0024] The seventh aspect of the present invention provides a method for producing the recombinant type II humanized collagen described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention, comprising the following steps:

[0025] (1) Culture the host cells described in the fifth aspect of the present invention under suitable culture conditions;

[0026] (2) Harvesting host cells and / or culture medium containing recombinant type II humanized collagen or fusion protein; and optionally...

[0027] (3) Purify recombinant type II humanized collagen or fusion protein.

[0028] The eighth aspect of the present invention provides a composition comprising one or more of the following: the recombinant type II humanized collagen of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the polynucleotide of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the host cell of the fifth aspect of the present invention, and the trimer collagen of the sixth aspect of the present invention.

[0029] In some embodiments, the composition is one or more of the following: bio-dressings, biomimetic materials, plastic or cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives; preferably, the composition is a surface composition, an injectable composition or an oral composition; preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge or fiber.

[0030] The ninth aspect of this invention provides the use of the recombinant type II humanized collagen described in the first aspect, the fusion protein described in the second aspect, the polynucleotide described in the third aspect, the carrier described in the fourth aspect, and the host cell described in the fifth aspect in the preparation of one or more of the following: bio-dressings, human biomimetic materials, plastic or cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives.

[0031] The tenth aspect of the present invention provides a method for promoting cell adhesion, the method comprising contacting cells with the recombinant type II humanized collagen of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the polynucleotide of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the host cell of the fifth aspect of the present invention, the trimer collagen of the sixth aspect of the present invention, and / or the composition of the seventh aspect of the present invention.

[0032] In some embodiments, the promotion of cell adhesion is performed in vitro.

[0033] The eleventh aspect of the present invention provides the use of recombinant type II humanized collagen in the preparation of products for promoting cartilage repair, wherein the recombinant type II humanized collagen comprises n repeating units, the repeating units being directly or connected by a linker, the repeating units comprising the amino acid sequence shown in any one of SEQ ID NO:1-3 or a variant thereof, the variant being (1) an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on any one of SEQ ID NO:1-3; or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-3.

[0034] In some implementations, n is an integer from 1 to 20; in other implementations, n is 1, 4, 8, or 12.

[0035] In some embodiments, the linker comprises one or more amino acid residues; preferably, the linker comprises 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0036] In some embodiments, the collagen comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:2 or SEQ ID NO:5.

[0037] In some embodiments, the collagen comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:5.

[0038] In some embodiments, the collagen comprises an amino acid sequence obtained by substitution, deletion, or insertion of one or more amino acids based on SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:5.

[0039] The advantages of this invention include:

[0040] 1. A novel core functional region of recombinant type II humanized collagen was provided, and a recombinant type II humanized collagen with good biological activity was successfully constructed based on the novel core functional region.

[0041] 2. The amino acid composition of the recombinant type II humanized collagen of the present invention is 100% identical to the corresponding part of the amino acid sequence of natural human type II collagen, and will not produce immune rejection or allergic reactions when applied to the human body.

[0042] 3. The recombinant type II humanized collagen of the present invention has excellent cell adhesion activity and can be used for cartilage repair.

[0043] 4. The preparation method of the present invention is simple and can produce high-yield recombinant type II humanized collagen on a large scale. Attached Figure Description

[0044] Figure 1 The electrophoresis results of recombinant humanized type II collagen II-1 are shown.

[0045] Figure 2 The electrophoresis results of recombinant humanized type II collagen II-2 are shown.

[0046] Figure 3 The electrophoresis results of recombinant type II humanized collagen II-3 are shown.

[0047] Figure 4 The CD diagram shows recombinant humanized collagen type II-1.

[0048] Figure 5 The CD diagram shows recombinant humanized collagen type II-2.

[0049] Figure 6 The CD diagram of recombinant type II humanized collagen II-3 is shown.

[0050] Figure 7 The attached diagram shows the cell adhesion of three recombinant type II humanized collagens.

[0051] Figure 8 Three recombinant type II humanized collagen proteins were shown to promote the proliferation of human chondrocytes C28 / I2.

[0052] Figure 9 Microscopic imaging of cell migration of three types of recombinant type II humanized collagen.

[0053] Figure 10 Cell migration rates of three recombinant type II humanized collagen proteins. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0055] Terminology Definition

[0056] As used in this article, "recombinant collagen" is a new type of biomaterial that uses cutting-edge structural biology, genetic engineering and other technologies to screen and prepare a gene encoding the functional region of human collagen specific type as a template, and obtains an amino acid sequence that is the same as or similar to that of human collagen.

[0057] As used herein, “recombinant type II humanized collagen” refers to a recombinant protein consisting of or substantially consisting of sequences derived from human type II collagen. In this context, recombinant type II humanized collagen may consist of or substantially consist of fragments or multiple repeats of fragments derived from human type II collagen.

[0058] As used herein, the term “expression” includes any step involved in peptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may contain the nucleic acids of this invention for introduction into cells for expression. Vectors may contain expression control elements operatively linked to the nucleic acids, such as promoters, terminators, and / or enhancers.

[0060] As used herein, the term "expression vector" refers to a straight or circular DNA molecule containing a polynucleotide encoding a polypeptide and operatively linked to a control sequence provided for its expression. In this paper, the expression vector is an *E. coli* expression vector.

[0061] As used herein, the term “recombinant expression vector” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain nucleic acid segments in a manner not normally found in nature, or is synthetic and contains one or more control sequences.

[0062] As used herein, the term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding region of the polynucleotide encoding the polypeptide.

[0063] As used herein, the term "host cell" refers to a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques. Host cells can be eukaryotic or prokaryotic cells. For example, eukaryotic cells include yeast cells, animal cells, and / or insect cells. Prokaryotic cells can be E. coli cells. Detailed Implementation

[0065] Recombinant Type II Humanized Collagen

[0066] This invention provides recombinant type II humanized collagen. The recombinant type II humanized collagen of this invention may comprise one or more repeating units, each repeating unit containing an amino acid sequence or a variant thereof shown in any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0067] In some embodiments, the variant sequence comprises an amino acid sequence obtained by mutating (substituting, adding, or deleting) one or more amino acid residues based on any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0068] In some embodiments, the variant sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the number of repeating units may be 1-20.

[0069] In some implementations, the number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0070] In some implementations, the number of repeating units is 1, 4, 8, or 12.

[0071] In some implementations, the mutation can be a substitution, such as a conserved amino acid substitution.

[0072] In some implementations, the repeating units are directly connected.

[0073] In some embodiments, each repeating unit is spaced apart by one or more (e.g., 2, 3, 4, 5, 6, 7, 8) amino acid residues.

[0074] In some implementations, when the repeating unit is mutated, the resulting recombinant collagen retains comparable or superior cell adhesion, cell proliferation activity, or cartilage repair capabilities compared to collagen obtained from the unmutated repeating unit.

[0075] In some embodiments, the recombinant type II humanized collagen of the present invention is synthetic or recombinantly expressed.

[0076] Recombinant type II humanized collagen encodes nucleic acid

[0077] This invention provides a polynucleotide encoding recombinant type II humanized collagen for expression.

[0078] In some embodiments, the polynucleotide is codon-optimized for the host cell in which it is expressed.

[0079] In some implementations, the polynucleotide encoding recombinant type II humanized collagen can be operatively linked to expression control elements, such as promoters, terminators, and / or enhancers, to form a nucleic acid or expression cassette.

[0080] In some embodiments, the polynucleotide encoding recombinant type II humanized collagen may also include nucleotides encoding purification tags, such as His tags, GST tags, MBP tags, SUMO tags, or NusA tags, or nucleotides encoding leader sequences to facilitate peptide purification or secretion.

[0081] Production methods

[0082] The present invention also provides a method for producing recombinant humanized type II collagen, comprising: (1) culturing host cells of the present invention under suitable culture conditions; (2) harvesting host cells and / or culture medium containing recombinant humanized type II collagen or fusion protein or fusion protein thereof; and (3) purifying said recombinant humanized type II collagen or fusion protein. The method of the present invention may include the step of enzyme digestion tag.

[0083] Compositions, kits and their applications

[0084] The recombinant type II humanized collagen of the present invention can be prepared into a composition or a kit.

[0085] In some embodiments, the composition or kit may be a composition or kit for tissue filling and / or volume enhancement.

[0086] In some embodiments, the composition or kit may also contain auxiliary substances.

[0087] In some embodiments, the compositions of the present invention may be cartilage repair agents comprising the polypeptides described herein. The compositions of the present invention may be injectable. The compositions of the present invention may be human structural materials, for example, suitable for cartilage repair, and do not elicit an immune response in the human body.

[0088] Construction and Production Method of Recombinant Type II Humanized Collagen

[0089] The present invention also provides a method for constructing recombinant type II humanized collagen, including functional region screening and strain construction, large-scale bio-fermentation, protein induction expression, purification and optional enzymatic digestion steps.

[0090] In some embodiments, the functional region screening and strain construction steps include: (1) large-scale functional region screening to obtain the target gene functional region; (2) inserting the obtained target gene functional region into an expression vector (e.g., PET-28a-Trx-His) to obtain a recombinant expression plasmid; (3) transforming the recombinant expression plasmid into competent Escherichia coli cells (e.g., BL21(DE3)) and screening to obtain positive Escherichia coli genetically engineered bacteria.

[0091] In some embodiments, the large-scale bio-fermentation step includes: adding the screened positive Escherichia coli genetically engineered bacteria to a shake flask containing antibiotic stock solution and culturing it in a constant temperature shaker at 220 rpm and 37°C.

[0092] In some embodiments, the steps of inducing protein expression include: (1) cooling the cultured shake flask to 16-30°C; (2) adding IPTG stock solution to induce expression; and (3) collecting the bacterial cells after inducing expression by placing the bacterial culture in a centrifuge bottle and centrifuging at 6000 rpm and 4°C for 12 min.

[0093] In some embodiments, the purification and optional enzymatic digestion steps of humanized type II collagen include: (1) crude purification of humanized type II collagen on a Ni affinity chromatography column; (2) enzymatic digestion with TEV enzyme in a certain proportion; and (3) purification of humanized type II collagen on an ion exchange column.

[0094] Example

[0095] The following embodiments are provided to illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.

[0096] Example 1: Construction and expression of recombinant type II humanized collagen

[0097] 1.1 Construction of recombinant type II humanized collagen

[0098] Large-scale functional region screening of natural human type II collagen (GenBank: EAW57970.1) yielded the following different protein functional regions.

[0099] GEQGPKGEPGPAGPQGAPGPAGEE(SEQ ID NO.1)

[0100] GSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPR(SEQ ID NO.2)

[0101] GFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLP(SEQ IDNO.3)

[0102] To ensure the purification and stability of recombinant type II humanized collagen, the amino acid fragments in these regions were optimized by repeated n times and directly linked to obtain recombinant collagen II-1, collagen II-2, and collagen II-3, with the corresponding amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:2, and SEQ ID NO:6, respectively.

[0103] The amino acid sequence of collagen II-1 (12 repeats):

[0104] GEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGE QGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEE(SEQ IDNO.4)

[0105] The amino acid sequence of collagen II-2 (single repeat):

[0106] GSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPR(SEQ ID NO.2)

[0107] The amino acid sequence of collagen II-3 (4 repeats):

[0108] GFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGF QGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLP(SEQ ID NO.5)

[0109] Codon optimization was performed using the E. coli expression system to obtain the coding nucleic acid sequences for collagen II-1, collagen II-2, and collagen II-3, as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0110] Collagen II-1 nucleic acid sequence:

[0111] GGAGAACAAGGGCCGAAAGGCGAACCGGGTCCGGCGGGTCCGCAGGGCGCACCGGGTCCGGCAGGCGAGGAAGGTGAGCAAGGTCCGAAAGGTGAGCCGGGTCCAGCTGGCCCTCAAGGCGCTCCGGGCCCGGCGGGCGAAGAAGGTGAGCAAGGCCCGAAGGGTGAGCCGGGTCCGGCCGGTCCGCAAGGCGCGCCAGGGCCGGCGGGTGAGGAAGGTGAGCAGGGTCCGAAAGGTGAGCCGGGTCCGGCGGGCCCGCAAGGTGCACCGGGCCCAGCTGGCGAAGAGGGCGAGCAGGGTCCGAAAGGCGAGCCGGGTCCGGCGGGCCCGCAGGGTGCGCCAGGCCCGGCGGGTGAAGAAGGCGAACAAGGCCCGAAAGGCGAACCGGGTCCGGCGGGTCCTCAAGGTGCCCCAGGCCCAGCGGGCGAAGAGGGCGAACAGGGTCCGAAGGGCGAGCCGGGGCCGGCGGGTCCGCAGGGCGCTCCGGGTCCGGCTGGCGAAGAAGGTGAACAGGGTCCGAAGGGCGAGCCGGGCCCGGCGGGTCCGCAAGGAGCACCGGGTCCGGCGGGCGAGGAAGGCGAACAGGGTCCCAAGGGTGAGCCGGGCCCGGCGGGTCCGCAAGGTGCCCCTGGCCCGGCGGGTGAAGAAGGTGAGCAGGGTCCCAAGGGCGAGCCGGGTCCGGCGGGTCCGCAGGGCGCACCGGGTCCGGCCGGCGAGGAGGGTGAACAAGGCCCAAAAGGCGAGCCGGGTCCGGCCGGCCCGCAGGGTGCTCCGGGTCCGGCTGGCGAAGAGGGCGAGCAGGGTCCGAAGGGTGAGCCGGGTCCGGCGGGCCCACAGGGCGCACCGGGTCCGGCCGGTGAGGAA(SEQ ID NO:6) Collagen II-2 nucleic acid sequence:

[0112] GGTAGCCCAGGTCCAGCAGGTCCGACAGGTAAACAAGGAGATCGTGGTGAAGCAGGAGCACAAGGACCAATGGGTCCAAGCGGTCCGGCAGGTGCAAGAGGTATTCAAGGGCCGCAAGGGCCACGCGGTGATAAAGGGGAAGCAGGTGAACCAGGTGAGAGAGGGTTAAAAGGACATCGTGGATTTACAGGACTGCAGGGTTTACCAGGTCCGCCGGGACCGAGCGGAGATCAAGGTGCAAGCGGTCCGGCGGGTCCGAGTGGTCCTCGT(SEQ ID NO:7)

[0113] Collagen II-3 nucleic acid sequence:

[0114] GGATTTCAGGGGTTGCCGGGCCCACCGGGTCCACCGGGCGAAGGTGGCAAGCCGGGCGACCAGGGCGTGCCGGGTGAGGCCGGTGCGCCAGGCCTGGTAGGTCCACGTGGTGAGCGCGGTTTCCCGGGTGAGCGCGGTTCTCCGGGTGCTCAAGGCCTGCAGGGTCCGCGTGGCCTGCCGGGCTTTCAGGGTTTGCCGGGCCCGCCGGGCCCGCCCGGCGAGGGTGGTAAGCCGGGCGACCAGGGCGTTCCGGGTGAAGCGGGTGCGCCAGGCCTGGTGGGCCCACGTGGTGAACGTGGTTTTCCGGGCGAACGCGGTAGCCCGGGCGCGCAGGGTCTGCAAGGACCGCGTGGCCTGCCGGGATTCCAGGGTCTCCCGGGTCCGCCTGGACCGCCGGGCGAAGGTGGCAAACCGGGTGATCAGGGTGTTCCGGGTGAAGCAGGCGCTCCGGGCCTGGTTGGTCCGCGTGGCGAACGTGGTTTTCCGGGCGAGCGCGGCTCCCCGGGTGCCCAAGGTTTACAAGGCCCGCGTGGTCTTCCGGGCTTCCAAGGCCTGCCGGGGCCGCCGGGCCCGCCTGGCGAGGGTGGTAAACCGGGCGATCAGGGTGTGCCGGGCGAGGCAGGTGCGCCGGGGTTGGTCGGTCCTAGAGGTGAACGTGGTTTCCCGGGTGAGCGCGGTAGCCCGGGTGCGCAAGGCCTGCAGGGCCCGCGTGGCCTGCCG(SEQ ID NO: 8)

[0115] 1.2 Expression of Recombinant Humanized Collagen Type II

[0116] The synthetic gene functional region was inserted into the pET-28a-Trx-His expression vector to obtain the corresponding recombinant expression plasmid.

[0117] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process was as follows: (1) The E. coli competent cells BL21(DE3) were taken out of the ultra-low temperature freezer and placed on ice. When they were half-thawed, 2 μl of the plasmid to be transformed was added to the E. coli competent cells BL21(DE3) and mixed slightly 2-3 times. (2) The mixture was placed on ice for 30 min, and then heat-shocked in a water bath at 42℃ for 45-90 s. After taking it out, it was placed on ice for 2 min. (3) It was transferred to a biosafety cabinet and 700 μl of liquid LB medium was added. Then it was cultured at 37℃ and 220 rpm for 60 min. (4) 200 μl of bacterial solution was evenly spread on LB plates containing kanamycin sulfate. (5) The plates were cultured in an incubator at 37℃ for 15-17 h until uniformly sized colonies grew.

[0118] Pick 5-6 single colonies from the transformed LB agar plates and place them in a shake flask containing antibiotic stock solution (ampicillin 100 mg / L). Incubate at 220 rpm and 37°C in a constant temperature shaker for a certain period of time until the colonies appear as a mist. Then, cool the shake flasks to 16-30°C, add IPTG (0.5 mM) to induce expression for a period of time, aliquot the bacterial culture into centrifuge bottles, centrifuge at 6000 rpm and 4°C for 12 min, collect the bacterial cells, record the cell weight, and perform electrophoresis analysis.

[0119] The collected bacterial cells were resuspended in a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). The bacterial suspension was cooled to ≤15℃ and homogenized twice or sonicated to disrupt the cells. After the cell disruption was completed, the bacterial suspension was collected. The disrupted bacterial suspension was aliquoted into centrifuge bottles and centrifuged at 17000 rpm and 4℃ for 30 min. The supernatant was collected.

[0120] The recombinant humanized type II collagen was purified and enzymatically digested. The specific process was as follows: (1) Crude purification: a. Equilibrate the column: Equilibrate the column with equilibration buffer (200mM sodium chloride, 25mM Tris, 20mM imidazole) at a flow rate of 10mL / min. b. Load the sample: Add the supernatant after centrifugation to the column until the liquid is completely discharged at a flow rate of 5mL / min. c. Wash away impurities: Add 100mL of washing buffer (200mM sodium chloride, 25mM Tris, 20mM imidazole) until the liquid is completely discharged at a flow rate of 10mL / min. d. Collect the target protein: Add 20 mL of elution buffer (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole), at a flow rate of 10 mL / min, and collect the flow-through. Detect the protein concentration using UV-Vis spectrophotometry. Calculate the protein concentration using the following formula (C(mg / ml) = A280 × dilution factor × extinction coefficient), and perform electrophoresis. e. Wash the column with 1 M imidazole working solution at a flow rate of 10 mL / min. (2) Enzyme digestion: Add TEV enzyme at a ratio of total protein to total TEV enzyme of 20:1, and digest at 16℃ for 2 h. Place the digested protein solution into a dialysis bag and dialyze at 4℃ for 2 h, then transfer to a new dialysis buffer (20 mM sodium chloride, 20 mM Tris) and dialyze overnight at 4℃. (3) Purification: a. Column equilibration: Equilibrate the column with solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 ml / min. b. Sample loading: Load the sample at a flow rate of 5 ml / min, collect the flow-through sample, and perform electrophoresis. Store the protein at 4°C. c. Elution: Wash the column with solution B (1 M sodium chloride, 20 mM Tris) for 5 CVs. d. Column washing.

[0121] SDS-PAGE was used to detect the purified recombinant protein. Electrophoresis results are shown below. Figures 1-3 .like Figures 1-3 It can be seen that collagen II-1, collagen II-2, and collagen II-3 were successfully expressed and prepared; at the same time, control collagen B1 (also derived from type II collagen) was prepared, and the amino acid and nucleic acid sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively:

[0122] GEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPPGPSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGANGIPGPIGPPGPRGRSGETGPA (SEQ ID NO: 9)

[0123] GGTGAACCAGGTCGTGAAGGTAGCCCAGGTGCAGATGGACCACCAGGTCGTGATGGTGCAGCAGGAGTGAAAGGAGATCGTGGTGAAACCGGTGCAGTAGGTGCACCTGGTGCGCCAGGTCCGCCAG GTAGCCCTGGTCCAGCCGGTCCTACCGGAAAACAAGGGGATAGAGGAGAAGCAGGAGCACAGGGTCCGATGGGTCCGTCAGGTCCGGCGGGTGCACGTGGTATTCAGGGTCCGCAGGGTCCGCGTGG TGATAAAGGTGAAGCAGGTGAACCGGGGGAAAGAGGATTAAAAGGGCATCGTGGTTTTACGGGTCTGCAGGGTCTGCCTGGTCCGCCTGGTCCGAGCGGTGATCAGGGTGCAAGCGGTCCGGCAGGT CCGAGCGGACCTCGTGGACCTCCGGGTCCTGTGGGTCCTAGTGGTAAGGATGGGGCAAATGGTATTCCTGGTCCTATTGGTCCGCCGGGTCCGCGTGGGAGATCAGGTGAAACCGGACCGGCA(SEQ ID NO:10)

[0124] After purification, the protein yield of collagen II-1 was 0.59%. The amount of the target protein after purification was relatively small. The theoretical molecular weight after purification was 25.9 KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight. Figure 1 The collagen II-2 protein yield was 0.55%, and the theoretical molecular weight after purification was 8.3 KD. The apparent molecular weight after purification was consistent with the theoretical molecular weight. Figure 2 Collagen II-3 was completely digested, resulting in high purity and expression levels after purification. The purification yield was 0.65%, and the theoretical molecular weight after purification was 22.4 KD. The apparent molecular weight after purification was consistent with the theoretical molecular weight. Figure 3 Collagen II-1, collagen II-2, and collagen II-3 were selected as target proteins for subsequent testing.

[0125] Example 2: Circular dichroism UV-Vis analysis of recombinant type II humanized collagen

[0126] Experimental methods

[0127] (1) Instrument parameter settings

[0128]

[0129] (2) Near and far ultraviolet scanning of standard samples

[0130] The scanning wavelength was set to 180-340 nm for background testing and blank buffer testing. Then, the circular dichroism near and far UV absorption of 1 mg / mL CSA standard solution in the 180-340 nm range was collected.

[0131] Sample processing

[0132] Take protein samples and concentrate them to a protein concentration of 1 mg / ml using a 10 kDa ultrafiltration concentrator (Millibo).

[0133] (4) Sample far-ultraviolet scanning

[0134] Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a far-ultraviolet scan at 190-260 nm according to the above parameters and collect the data.

[0135] (5) Near-ultraviolet scanning of samples

[0136] Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a near-ultraviolet scan at 250-340 nm according to the above parameters and collect the data.

[0137] (6) Scanning image processing

[0138] All scanned spectra were processed using the software Pro-Data Viewer to extract baselines and smooth out the images.

[0139] The circular dichroism UV-Vis analysis results of recombinant collagen II-1, II-2, and II-3 are as follows: Figure 4 , Figure 5 and Figure 6 As shown, the expression of recombinant type II humanized collagen showed positive peaks at 220-230 nm, indicating that collagen II-1, II-2, and II-3 all have triple helix structures.

[0140] Example 3: Bioactivity of recombinant humanized type II collagen

[0141] The method for detecting collagen activity can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0142] (1) The concentration of the protein samples to be tested was detected using ultraviolet absorption, including bovine type I collagen (China National Institutes for Food and Drug Control, No.: 380002), collagen II-1, collagen II-2, collagen II-3 provided in this invention, and control collagen B1. The ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated using the empirical formula C (μg / mL) = 144 × (A215 - A225), where C represents the protein concentration in the sample to be tested, A215 represents the absorbance of the sample to be tested at a wavelength of 215 nm, and A225 represents the absorbance of the sample to be tested at a wavelength of 225 nm. Note that the detection must be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far-ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues that do not develop color in Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.) After detecting the protein concentration, adjust the concentration of all test proteins to 0.5 mg / mL with PBS.

[0143] (2) Add 100 μL of various protein solutions and blank PBS solution control (NC) to a 96-well plate and let it stand at room temperature for 60 min.

[0144] (3) Add 10 to each hole 5 A well-cultured 3T3 / NIH cell was incubated at 37°C for 60 minutes.

[0145] (4) Wash each well with PBS 4 times.

[0146] (5) The absorbance at OD450nm was measured using the CCK8 assay kit (Beyotime, product catalog number C0038). The cell adhesion rate could be calculated based on the values ​​of the blank control. The calculation formula is as follows: Cell adhesion rate = {(test wells - blank wells) / (positive wells - blank wells)} × 100%. The cell adhesion rate reflects the activity of collagen. The higher the protein activity, the better it can provide a high-quality external environment for cells in a short time, helping cells adhere.

[0147] The results are as follows Figure 7 As shown in the comparison, it can be seen that compared with the positive control (PC) bovine type I collagen (B ColI group, 0.5 mg / ml) and control collagen B1, the humanized collagen II-1, II-2 and II-3 of the present invention have superior bioadhesion activity.

[0148] Example 4: The cartilage repair effect of recombinant humanized type II collagen

[0149] 4.1. Cell proliferation experiment

[0150] The assay method for detecting the cell-promoting activity of collagen can refer to the People's Republic of China Pharmaceutical Industry Standard YYT1849-2022, Recombinant Collagen. The specific implementation method is as follows:

[0151] (1) The principle of this method is as follows: CCK8 reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan dye under the action of electron carrier 1-methoxy-5-methylphenazine sulfate dimethyl ester (1-Methoxy PMS). The amount of formazan dye generated is directly proportional to the number of live cells. Therefore, this property can be used to directly analyze cell proliferation.

[0152] (2) Experimental grouping: DMEM complete culture medium was used as negative control, Native Human Collagen II protein (Abcam, #ab134522) was used as positive control, collagen II-1, II-2, and II-3 solutions were used as test samples, and complete culture medium without cells was used as zeroing well.

[0153] (3) Sample preparation: Dissolve collagen in complete culture medium to the highest concentration used, filter sterilize with 0.22μm microporous membrane, and prepare samples of each concentration.

[0154] (4) Cell plating: When the confluence of human chondrocyte C28 / I2 cells in the T75 culture flask reaches approximately 90%, trypsin digestion and counting are performed. Dilute 1 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates, and 100 μL of PBS was added to the edge wells to block adhesion. The cells were cultured for 24 hours.

[0155] (5) Cell drug delivery: After the cells adhered to the wall, the supernatant was aspirated and the corresponding solutions were replaced in sequence. Each group had 4 replicates and were cultured for 48 hours.

[0156] (6) Experimental detection: Discard the culture medium, add 100 μL (including 5 μL CCK8 solution) of basal culture medium, incubate in an incubator for 1-2 h, and detect using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The cell viability calculation formula is as follows.

[0157] Cell viability = {(As-Ab) / (Ac-Ab)} × 100%.

[0158] Where As is the absorbance of the recombinant collagen test well, Ab is the absorbance of the zeroing well, and Ac is the absorbance of the negative well.

[0159] The above methods were used to detect collagen II-1, collagen II-2, collagen II-3, control collagen, and bovine serum albumin (BSA) (Beyotime catalog number: ST023, irrelevant protein control). Figure 8 The results showed that, compared with the control group, collagen II-1, collagen II-2, and collagen II-3 had a significant proliferative effect on human chondrocyte C28 / I2.

[0160] 4.2 Cell migration assay

[0161] (1) Human chondrocytes C28 / I2 were cultured in 48-well plates. After the cells were confluent, a 10 μL pipette tip was used to create a uniform scraping wound on the monolayer of cells. The cells were washed with sterile PBS 1-2 times to ensure that there were no cell debris or suspended cells remaining, which would affect subsequent experiments.

[0162] (2) Add 1 mg / mL of collagen II-1, collagen II-2, collagen II-3 and control collagen (Abcam, #ab134522). Cells in the untreated culture medium group were used as blank controls. Observe the scratch closure at 0, 6 and 12 hours.

[0163] (3) Calculate the scratch area. Obtain the percentage of scratch closure using the following formula:

[0164] Scratch closure rate (%) = (A0-At) / A0×100%, where A0 is the scratch area at 0h and At is the scratch area at a specified time.

[0165] Cell migration experiments were used to verify their migration ability in chondrocytes. Collagen II-1 and collagen II-2 showed stronger proliferative capacity in chondrocytes, both reaching closure after 12 hours, while collagen II-3 showed slightly weaker proliferative activity in chondrocytes. Figure 9-10 It is evident that the scratch closure rate increased with treatment duration under the action of the three types of collagen II. At a treatment time of 12 hours, the scratch closure rates of the three type II collagens were significantly higher than those of the control collagen. In summary, collagen II-1, collagen II-2, and collagen II-3 of this invention all possess excellent repair and healing capabilities; within a 12-hour timeframe, they showed a significant promoting effect on chondrocyte migration, a difference that was highly pronounced compared to the control collagen. The three types of collagen II of this invention have significant potential application value and prospects in promoting chondrocyte regeneration, enhancing cell migration ability, and treating diseases such as osteoarthritis.

Claims

1. A recombinant humanized collagen type II, characterized in that, consisting of n directly connected repeat units, the amino acid sequence of the repeat units being one of SEQ ID NO: 1 or SEQ ID NO: 3, n being an integer from 4 to 20, the recombinant humanized collagen type II having a cell adhesion activity.

2. The recombinant humanized collagen type II according to claim 1, wherein n is 4, 8 or 12.

3. The recombinant humanized collagen type II according to claim 1, the amino acid sequence of the collagen being SEQ ID NO: 4 or SEQ ID NO:

5.

4. A fusion protein consisting of the recombinant humanized collagen type II according to any one of claims 1 to 3 and a purification tag and / or a leader, the leader being a synthetic leader sequence, a signal peptide or a leader peptide.

5. The fusion protein according to claim 4, wherein the purification tag is selected from a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag.

6. A polynucleotide encoding the collagen according to any one of claims 1 to 3 or the fusion protein according to claim 4 or 5.

7. The polynucleotide according to claim 6, comprising the nucleotide sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 8 or a degenerate sequence thereof.

8. A vector comprising the polynucleotide according to claim 6 or 7.

9. The vector according to claim 8, which is an expression vector.

10. The vector according to claim 8 or 9, comprising a control element operably linked to the polynucleotide.

11. The vector according to claim 10, wherein the control element is selected from a promoter, a terminator and / or an enhancer.

12. A host cell comprising the polynucleotide according to claim 6 or 7 or the vector according to any one of claims 8 to 11.

13. The host cell according to claim 12, which is a bacterial, a fungal or an animal cell.

14. The host cell according to claim 13, wherein the bacterial is E. coli; the fungal is a yeast.

15. The host cell according to claim 14, wherein the yeast is S. cerevisiae.

16. A trimeric collagen protein, which is a trimeric collagen protein of the recombinant humanized collagen type II according to any one of claims 1 to 3.

17. A method of producing the recombinant humanized collagen type II according to any one of claims 1 to 3 or the fusion protein according to claim 4 or 5, characterized in that, comprising the following steps: (1) culturing the host cell according to any one of claims 12 to 15 under suitable culture conditions; (2) harvesting the host cell and / or the culture medium comprising the recombinant humanized collagen type II or the fusion protein; and optionally (3) purifying the recombinant humanized collagen type II or the fusion protein.

18. A composition characterized in that, one or more of the recombinant humanized collagen type II according to any one of claims 1 to 3, the fusion protein according to claim 4 or 5 and the trimeric collagen protein according to claim 16.

19. The composition according to claim 18, which is one or more of a biological dressing, a human bionic material, an orthopedic or cosmetic material, an organoid culture material, a coating material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a 3D-printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

20. The composition according to claim 19, wherein the composition is a surface composition, an injection composition, or an oral composition.

21. The composition according to claim 19, wherein the composition is a solution, a lyophilized powder, a gel, a sponge, or a fiber-form composition.

22. The composition according to claim 19, wherein the human bionic material is a cardiovascular stent material.

23. The composition according to claim 19, wherein the orthopedic or cosmetic material is a tissue injection filling material.

24. Use of the recombinant humanized collagen type II according to any one of claims 1 to 3, the fusion protein according to claim 4 or 5, the polynucleotide according to claim 6 or 7, the vector according to any one of claims 8 to 11, the host cell according to any one of claims 12 to 15, and / or the trimeric collagen according to claim 16, for the manufacture of one or more of a biological dressing, a human bionic material, an orthopedic or cosmetic material, an organoid culture material, a coating material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a 3D-printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

25. The use according to claim 24, wherein the human bionic material is a cardiovascular stent material.

26. The use according to claim 24, wherein the orthopedic or cosmetic material is a tissue injection filling material.

27. An in vitro method of promoting cell adhesion, characterized in that, The method comprises contacting a cell with the recombinant humanized collagen type II according to any one of claims 1 to 3, the fusion protein according to claim 4 or 5, the trimeric collagen according to claim 16, and / or the composition according to any one of claims 18 to 23.

28. Use of the recombinant humanized collagen type II according to any one of claims 1 to 3, for the manufacture of a product for promoting cartilage repair.

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