Collagen and its uses

Abstract

The present invention provides collagen and its use. The collagen includes an N-terminal sequence and a C-terminal sequence. The N-terminal sequence includes one or more repeating units, and the repeating unit includes the amino acid sequence shown in SEQ ID NO. 15. The recombinant type II humanized collagen produced by the present invention has high activity in promoting cell adhesion, does not cause an immune reaction when applied to the human body, can obtain recombinant type II humanized collagen on a large scale, and is expected to be widely applied in the field of cartilage repair.

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application filed on May 12, 2023, bearing application number 202310537499.9 and entitled "Polypeptide and Use Thereof," the contents of which are incorporated herein by reference.

[0002] The present invention belongs to the technical field of synthetic biology, and specifically relates to human anatomical materials and methods for their biosynthetic production. [Background technology]

[0003] Collagen is a type of protein that is widely distributed in the connective tissues of the human body and is the most abundant protein in the human body, accounting for 25% to 35% of the total protein. At present, it has been found that there are at least 28 collagen subtypes in the human body, each located in different tissues and organs.

[0004] Type II collagen is a type of high molecular weight protein found mainly in cartilage tissue, vitreous body, and cornea. Its thread-like collagen fibers are intertwined with elastin and polysaccharide proteins to form a network structure, also known as complex collagen. Type II collagen is a necessary component for cartilage and bone formation, bone growth, and the maintenance of mature cartilage, so undenatured collagen can be a structural and functional component of cartilage.

[0005] Type II collagen, a major component of articular cartilage matrix, protects cartilage from wear, along with lubricating components such as hyaluronic acid and proteoglycans. With aging, the rate of collagen synthesis gradually declines, resulting in increased collagen loss, degenerating articular cartilage, increasing interbone friction, and causing joint inflammation. Currently, the most common treatment for osteoarthritis involves ingesting collagen, but its protein utilization rate is low, slowing the cartilage regeneration rate, which is detrimental to patient rehabilitation and impacts the process and efficiency of treatment. Due to the poor therapeutic efficacy of current treatments, patients ultimately have no choice but to undergo artificial joint replacement surgery, which is not only expensive but can also lead to fatal complications such as postoperative blood clot formation and postoperative infection, resulting in limb amputation.

[0006] However, collagen materials currently used as cartilage are mainly derived from extraction of animal cartilage, which does not eliminate the immunogenicity of animal-derived collagen, and the impurity removal and extraction processes are still complicated, making it difficult to ensure the triple helix structure of collagen, making it unsuitable for large-scale production.

[0007] At the same time, the immune response of animal-derived collagen is also an important factor limiting the application of collagen. With the growing expansion of China's collagen industry, the use of biosynthetic pathways to obtain collagen is becoming increasingly mature, with humanized collagen in particular taking the lead in the world. In 2021, the National Medical Products Administration of China established a nomenclature and classification for biosynthetic collagen, stating that recombinant humanized collagen refers to the full-length or partial functional domain of the amino acid sequence encoded by a specific human collagen gene produced through DNA recombinant technology, or a combination of functional domains containing human collagen functions.

[0008] Traditional collagen production methods involve treating animal tissues with acid, alkali, or enzymatic hydrolysis to extract collagen derivatives. However, the collagen extracted by these methods loses its original biological activity and cannot function in biomedical applications. With the development of modern technology, several extraction methods have emerged in China and abroad that involve removing impurities from animal cartilage and subjecting it to enzymatic hydrolysis to obtain undenatured type II collagen. While the resulting collagen can be used for cartilage repair, these methods require long removal and extraction times, resulting in low product purity and poor product stability, making them unsuitable for mass production. To address the shortcomings of the prior art, one company proposed using Pichia yeast as a host strain to produce type II collagen for cartilage repair. However, because this collagen is not humanized, it has a certain immunogenicity.

[0009] Therefore, there is a strong need for recombinant type II humanized collagen that can be directly injected into the human body and does not induce immunogenic reactions, for application in cartilage repair as a structural material in the human body. Summary of the Invention [Problem to be solved by the invention]

[0010] Human structural materials mainly contain structural proteins in collagen, which have complex structures and precise functions and are of great significance for the repair and regeneration of human tissues, but are not easily obtained by conventional production methods. The present invention utilizes synthetic biology and structural biology techniques to develop a high molecular weight functional protein that has the triple helix structure of human type II collagen and can function as human collagen. The inventors conducted a large-scale screening of functional regions. First, they performed sequence screening to eliminate regions that did not contain these important charge motifs, since the Gly-XY repeat sequence in the helical region of native type II collagen contains a large number of charged amino acids, and these charges interact to bind to cells. Second, they used a computer-aided protein structure prediction method to screen for potential helical functional regions with the most interchain hydrogen bond structures and the most stable trimer aggregation. Third, they used a protein expression property prediction method to screen for functional regions of human type II collagen that have the highest protein expression, are easy to purify, and are highly stable. Fourth, they optimized the direct linkage of amino acid fragments from these regions by repeating them n times (the repetition was performed to ensure that the molecular weight of the recombinant humanized type II collagen was within a certain range, allowing it to be easily purified and stabilized).

[0011] The present invention includes screening, synthesis process and application scenarios of the core functional domain of recombinant type II humanized collagen. The design of the present invention is the first to invent the screening and protein synthesis process of the functional domain of recombinant type II humanized collagen, which can be directly injected into the human body and used for cartilage repair.

[0012] The present invention is based in part on the inventors' unexpected findings: Compared to other recombinant type II humanized collagens, recombinant type II humanized collagen HC2B-A10 is expressed in higher amounts upon recombinant expression, is easier to isolate and purify, and has a stronger cell adhesion effect than bovine type I collagen. [Means for solving the problem]

[0013] In one aspect, the collagen or polypeptide of the present invention comprises a plurality of repeat units comprising the amino acid sequence shown in SEQ ID NO. 15 (gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak) or a variant having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein each repeat unit is directly linked or separated by one or more amino acid residues. In one embodiment, the repeat unit may be an amino acid sequence obtained after the amino acid sequence of SEQ ID NO. 15 has undergone mutation (substitution, insertion, deletion, or addition) of one or more amino acid residues.

[0014] In one embodiment, the number of repeating units is 1 to 20. In one embodiment, the number of repeating units is 6 to 10. In one embodiment, the number of repeating units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0015] In one embodiment, the collagen or polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 10. In one embodiment, the collagen or polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 10, an amino acid sequence obtained after the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 10 has undergone mutation (substitution, insertion, deletion, or addition) of one or more amino acid residues, or a variant having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO. 10. In one embodiment, when the collagen or polypeptide sequence is mutated, the resulting collagen or polypeptide retains the functions of the present invention, such as cell adhesion and cartilage repair capabilities.

[0016] In one aspect, a polynucleotide according to the invention encodes a collagen or polypeptide as described herein.

[0017] In one embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.29.

[0018] In one aspect, a nucleic acid of the invention comprises a polynucleotide described herein. Optionally, the nucleic acid further comprises nucleotides encoding a purification tag, such as a His tag, a GST tag, a MBP tag, a SUMO tag, or a NusA tag. Optionally, the nucleic acid further comprises nucleotides encoding a leader sequence.

[0019] In one aspect, a vector according to the invention comprises a polynucleotide or nucleic acid as described herein.

[0020] In one embodiment, the vector is an expression vector. In one embodiment, the vector comprises an expression control element, such as a promoter, terminator, and / or enhancer, operably linked to the polynucleotide or nucleic acid.

[0021] In one aspect, a host cell of the present invention comprises a polynucleotide, nucleic acid, or vector described herein. In one embodiment, the host cell is a bacterial, fungal, or animal cell. In one embodiment, the bacterium is Escherichia coli. In one embodiment, the fungus is a yeast, such as Saccharomyces cerevisiae.

[0022] In one aspect, the method of the present invention for producing collagen or a polypeptide described herein comprises: (1) culturing a host cell described herein under suitable culture conditions; (2) harvesting the host cells and / or medium containing the collagen or polypeptide; and (3) purifying the collagen or polypeptide.

[0023] In one aspect, the composition of the present invention comprises collagen or a polypeptide as described herein. In one embodiment, the composition is a kit. In one embodiment, the composition is one or more of the following: bio-coating materials, human biomimetic materials, cosmetic materials, organoid culture materials, cardiovascular stents, 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 printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical auxiliary materials, and food additives, and is preferably an injectable composition or an oral composition. In one embodiment, the composition is an injectable composition or an oral composition.

[0024] In one embodiment, the composition is a composition for cartilage repair. In one embodiment, the composition is an injectable composition for cartilage repair.

[0025] In one aspect, a method of the present invention for enhancing cell adhesion comprises contacting a cell with a collagen or polypeptide described herein, a polynucleotide described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, and / or a composition described herein.

[0026] In one aspect, the present invention provides use of a collagen or polypeptide described herein, a polynucleotide described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, and / or a composition described herein in the manufacture of a kit for enhancing cell adhesion or cartilage repair. In one aspect, the present invention provides use of a collagen or polypeptide described herein, a polynucleotide described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, and / or a composition described herein in one or more of a bio-covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection and filling material, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D-printed artificial organ biomaterial, a cosmetic raw material, a medicinal supplement, and a food additive.

[0027] Advantages of the present invention include:

[0028] 1. The present invention provides the core functional region and amino acid sequence of recombinant humanized type II collagen. 2. This invention is the first to successfully synthesize recombinant humanized type II collagen that can be injected into the human body and used for cartilage repair. 3. The amino acid composition of the produced recombinant type II humanized collagen is 100% identical to the corresponding part of the amino acid sequence of natural collagen, and does not cause immune rejection or allergic reactions when applied to the human body. 4. The manufacturing method described herein is simple and allows for the large-scale production of high yields of recombinant humanized type II collagen. 5. Recombinant type II humanized collagen HC2B-A10 achieves optimal expression, isolation and purification effects compared with other recombinant type II humanized collagens. 6. The recombinant type II humanized collagen of the present invention has better biological adhesive activity, i.e., recombinant type II humanized collagen HC2B-A10>bovine type I collagen. [Brief explanation of the drawings]

[0029] [Figure 1] 1 shows the crude purification status of recombinant type II humanized collagen HC2B-a6 and HC2B-b5. [Figure 2] 1 shows the crude purification status of recombinant type II humanized collagen HC2B-b1, HC2B-a3, and HC2B-b3. [Figure 3] 1 shows the purification status of recombinant type II humanized collagen B3Q, HC2B-A8, and HC2B-A7. [Figure 4] The purification status of recombinant type II humanized collagen egg HC2B-A10 and the crude purification status of HC2B-B9. [Figure 5] 1 shows the purification status of recombinant type II humanized collagen HC2B-A9. [Figure 6] 1 shows the crude purification status of recombinant type II humanized collagen HC2B-B8, HC2B-B7, and HC2B-B10. [Figure 7] 1 shows the refinement status of recombinant type II humanized collagen HC2B-B7 and HC2B-B9. [Figure 8] 1 shows the activity detection status of recombinant type II humanized collagen HC2B-A10. [Figure 9] 1 is a map of the pET-28a-Trx-His expression vector. DETAILED DESCRIPTION OF THE INVENTION

[0030] In order to clarify the purpose, technical means and advantages of the present invention, the following will clearly and completely describe the technical means in the embodiments of the present invention with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments that a person skilled in the art can come up with without any creative efforts fall within the protection scope of the present invention.

[0031] Recombinant collagen is produced by screening and manufacturing using advanced structural biology, genetic engineering and other technologies, using the genetic code of the functional domain of a specific type of human collagen as a template, to obtain novel biomaterials with amino acid sequences identical or similar to those of human collagen.

[0032] As used herein, a "polypeptide" is a plurality of amino acid residues linked via peptide bonds. Collagen, recombinant collagen, recombinant type II humanized collagen, or polypeptide are used interchangeably herein.

[0033] As used herein, collagen or polypeptide may comprise one or more repeat units comprising the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence obtained after the amino acid sequence has undergone mutation (substitution, addition, insertion, or deletion) of one or more amino acid residues. The number of repeat units is 1 to 20. For example, the number of repeat units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In particular, the mutation may be a substitution, for example, a conservative amino acid substitution. The amino acid sequence of SEQ ID NO. 15 is gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak. Each repeat unit may be directly linked or separated by one or more amino acid residues.

[0034] When the collagen or polypeptide sequence is mutated or has a spacer sequence, the resulting collagen or polypeptide retains the functions of the present invention, such as cell adhesion, cartilage repair ability, etc.

[0035] The collagen or polypeptide of the present invention may be synthesized or expressed recombinantly. In the case of recombinant expression, the collagen or polypeptide of the present invention may be encoded by a polynucleotide. The polynucleotide may be codon-optimized for the host cell in which it is expressed. The polynucleotide encoding the collagen or polypeptide may be operably linked to expression control elements, such as a promoter, terminator, and / or enhancer, to form a nucleic acid or expression cassette. The nucleic acid may further contain nucleotides encoding a purification tag, such as a His tag, GST tag, MBP tag, SUMO tag, or NusA tag, or nucleotides encoding a leader sequence, to facilitate purification or secretion of the collagen or polypeptide.

[0036] As used herein, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide can be inserted. A vector is called an expression vector if it can express a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, allowing the genetic material elements carried therein to be expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages, such as lambda phage or M13 phage, and animal viruses. A vector can contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector can further contain a replication origin. A vector can contain a nucleic acid of the present invention for introduction into a cell and expression. The vector can include expression control elements, such as a promoter, terminator, and / or enhancer, operably linked to the nucleic acid.

[0037] As used herein, the term "host cell" refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA accelerated by electroporation, lipid transfection, and particle guns. Host cells may be eukaryotic or prokaryotic cells. For example, eukaryotic cells are yeast cells, animal cells, and / or insect cells. Prokaryotic cells may be E. coli cells.

[0038] The present invention further provides a method for producing collagen or polypeptide, comprising the steps of (1) culturing the host cells herein under suitable culture conditions, (2) harvesting the host cells and / or medium containing the collagen or polypeptide, and (3) purifying the collagen or polypeptide. The method of the present invention can include a step of enzymatic cleavage of the tag.

[0039] The collagen or polypeptide of the present invention can be prepared into a composition or kit. The composition or kit may be a composition or kit for tissue filling and / or compatibilization. The composition or kit may further comprise an auxiliary substance. The composition of the present invention may be a cartilage repair agent comprising the collagen or polypeptide described herein. The composition of the present invention may be injectable. The composition of the present invention may be a human structural material that can be used, for example, for cartilage repair and does not provoke an immune response in the human body.

[0040] As used herein, the relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity." For purposes of the present invention, sequence identity between two amino acid sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or an updated version. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the unabbreviated option) is used as the percentage identity, calculated as follows:

[0041] (Identical residues × 100) / (alignment length − total number of gaps in the alignment) For purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, as above) implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, as above), preferably version 5.0.0 or an updated version. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the unabbreviated option) is used as the percentage identity, calculated as follows:

[0042] (identical deoxyribonucleotides × 100) / (alignment length − total number of gaps in the alignment) As used herein, certain mutations may be present in the repeating units or collagens or polypeptides of the present invention. For example, the amino acid sequence of one or more of these portions may include substitutions, deletions, additions, or insertions of amino acid residues. In the context of amino acid mutations, "multiple" may refer to 2 to 40, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, or 39, or any range therebetween.

[0043] That is, the present invention allows for the use of repeat unit variants as long as they have the activity of promoting cell adhesion. Specifically, the variants may have a certain percentage of identity with a specific sequence (any collagen or polypeptide sequence described herein), e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The specific sequence may be any sequence of the present invention, e.g., SEQ ID NOs. 1-16, but it is preferable that these variants retain their intended function, such as enhancing cell adhesion or cartilage repair.

[0044] The method of the present invention comprises: In light of the current state of research, the present invention provides a method for biosynthesizing recombinant humanized type II collagen, i.e., a method for producing a human body structural material. The method may include one or more of the following steps: (1) screening for functional regions and constructing a strain; (2) large-scale biological fermentation culture and inducible protein expression; and (3) purification and optional enzymatic cleavage of the humanized type II collagen.

[0045] Screening for functional regions and constructing strains can be carried out as follows: (1) large-scale screening for functional regions to obtain the target gene functional region; (2) inserting the obtained target gene functional region into the PET-28a-Trx-His expression vector to obtain a recombinant expression plasmid; (3) introducing the recombinant expression plasmid into E. coli competent cells BL21(DE3) and screening to obtain positive E. coli genetically engineered strains.

[0046] Large-scale biofermentation can be carried out as follows: Positive E. coli genetically engineered strains obtained by screening were added to the antibiotic stock solution in shake flasks and cultured in a constant temperature shaker at 220 rpm and 37°C.

[0047] Induction of protein expression can be carried out as follows: (1) After cultivation, the shake flask was cooled to 16-30°C, (2) IPTG mother liquor was added to induce expression, and (3) the bacterial solution after induction of expression was placed in a centrifuge flask and centrifuged at 6000 rpm at 4°C for 12 minutes, after which the bacterial cells were collected.

[0048] Purification and optional enzymatic cleavage of humanized type II collagen can be performed as follows: (1) crude purification of humanized type II collagen using a Ni affinity chromatography column, (2) enzymatic cleavage by adding TEV enzyme at a certain ratio, and (3) precision purification of humanized type II collagen using an ion exchange column.

[0049] The functional regions screened are shown below. (1) The amino acid sequence of HC2B-a3: gkpgddgeagkpgkagergppgpqgargfgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagargndgqp (SEQ ID NO: 1), (2) The amino acid sequence of HC2B-a6: gkpgkagergppgpqgargfgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfgtpglpgvkghrgkpgkagergppgpgvkghrgkpgvkghrgkpgkagppgpqgargfpgtpglpgvkgrgkpgkpgkagerg (SEQ ID NO:2) , (3), Amino acid sequence of HC2B-b1: gepgregrespgadgppgrdgaagvkgdrgetgavgappgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpagpsgppgppgppgvgpsgkdgangipgpigppgprgrsgetgpa (SEQ ID NO:3) , (4), HC2B-b3 amino acid sequence: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO:4) , (5), Amino acid sequence of HC2B-b5:gargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgqgprgdkgeagopgepgppgargiqgpgpglkghrgftglqglpgppgpglkghrgftglqgpgppgpglkghrgftglqgppgppgpgpsgargiqgpgqgpprgdkgeagopgepglkghrgftglqglpgppgppgppgppgppgpgppgpsgargiqgpgqgppgppgpgps (SEQ ID NO:5), (6), The amino acid sequence of B3Q: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpgqgprgdkgeagepgerglkgghrgftglqglpgppgpsgdqg asgpagpsgpsggpgpagptgkqgdrgagaqgrgargiqgpgqgrgdkgeagepgerglkgggggghrgftglqglqglpgppgpsgpsgdqgas gpagpgptgkqgdrgagarggaqgpsgpstgkqgargiqgqgpqgpgqgprgqgprgdkgepgepglkghftglqgppgpsgpsgdqgqgqgas (SEQ ID NO:6) , (7), Amino acid sequence of HC2B-A7: gpqgargfpgtpglpgvkghrgypgldgakgpqgargfgtpglpgvkghrgypgldgakgpqgargfpggtpglpgvkghrgypgldgakgpqgargfgtpglpgvkghrgypgldgakgpqgargfgtpglpgvkghrgypgldgakgpqgarggfgtpglpgvkghrgypgldgakgpqgarggtpglpgvkghrgypgldgakgqgarggfgtpglpgvkghrgypgldgakgpqgldgakgldgakgpqgldgakgpgpglpgvkghrgypgldgak (SEQ ID NO:7) , (8), The amino acid sequence of HC2B-A8: gargfpgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakghrgfgggldgakghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgldgakgargfgtpglpgvkgggghrgdgakgldgak (SEQ ID NO:8), (9), The amino acid sequence of HC2B-A9: gfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpglpgvkghrgypgldgakgldgakgfpgtpglpgvkghrgypgldgakgfpgvgghrgypgldgakgfgtpglpgvkghrgypgldgakgldgakgfgtpglpgvkghrgypgldgakgfgtpgvkghrgypgldgakgldgakgfpglpgvkghrgypgldgakgvkghrgypgldgakgldgakglpgvkghrggypgldgakgfglpgvkghrggggfgtpgvgrggdgakglpgldgakgpgldgakgldgak (SEQ ID NO: 9), (10), the amino acid sequence of HC2B-A10: gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpgvkgghrgypgldgakgtpglpgvkghrgypgldgakgldgakgtpgvkgghrgypgldgakgldgakgtpgvkghrgypgldgakglpgvkghrgypgldgakgtpgvkghrgypgldgakgldgakgtpglpgvkghrggdgakgypgldgakgldgakgtpglpgvkghrggypgldgak (SEQ ID NO: 10) , (11), the amino acid sequence of HC2B-B7: gerglkghrgftglqglpgppgpsgdqgasgpagpsgprgglkghrgftglqglpgppgpsgdqgasgpagpsgprggpglkghrgftglqglpgppgpsgpsgdqgasgpgpgpsgpsgdqgasgpgpgpglkghrgftglqglpgppgppgpsgdqgasgpgpgpgpsgrgftglqglpgpgpsgpgpsgpsgdqgasgpgpglkghrgftglqglqgpgpgppgpsgdqgasgpgpgpsgpr (SEQ ID NO: 11), (12), The amino acid sequence of HC2B-B8: gerglkghrgftglqglpgppgpsgdqgasgpaglkghrgftglqglpgppgpsgdqgasgpaglkghrgftglqglpgpgpgpgdqgasgpglkghrgftglqglpgppgpsgdqgasgpaglkghrgftglqglpgppgpsgdqgasgpaglkghrgft glqgpgpgpgpgpgdqgasglkghhrgftglqgpgppgpgpgpgpgpggpgftglqgpgppgpgpgpsgdqgasgpg lkghrgftglqglpgppgpgpsgdqgasgpgpgpgpgpgftglqglkghrgftglqgpggppgpgpgdqgasgpgpa (SEQ ID NO:12) , (13), the amino acid sequence of HC2B-B9: gerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglqglpgpgppgpsgdqgerglkghrgftglqglqglpgpgppgpsgdqgerglkghrgftglqglqglpgpgppgpsgdqglkghrgftglqglpggppgpsggdqgerglkrgftglqglpggggpsgfglqgppgpsgdqgerglkghrgftglqglqgpgpgpgpsgdqggghqgppgpgpsgdqgppgppgppgpsgdqgppgppgppgpsgdqglkgrgftglqglpggggpsgdq (SEQ ID NO:13) , (14), the amino acid sequence of HC2B-B10: gerglkghrgftglqglpgppgglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgppgerglkghrgftglqglpgppgppgerglkghrgftglqglpgppglkghrgftglqglpgppglkghrgftglqgppglkghrgftglqgpgppglkghrgftglqgpgppglkgftglqgpgppgppgppglkghrgftglqglkghrgftglqgpgpp (SEQ ID NO:14) .

[0050] The recombinant type II humanized collagen amino acid sequence produced in the present invention can be derived from the functional region of natural human type II collagen, and includes proteins in which the functional region and similar functional regions and amino acid sequences are mutated and modified, respectively.

[0051] The recombinant type II humanized collagen produced in the present invention is completely identical to the amino acid sequence of human collagen, and therefore can be directly injected into the human body for cartilage repair without causing immunogenic reactions.

[0052] Collagen or polypeptide of the present invention The present invention provides collagens or polypeptides comprising the amino acid sequence of any one of SEQ ID NOs. 1 to 14 or an amino acid sequence in which the amino acids are mutated. The mutation may be a substitution, addition, deletion, or insertion. Preferably, the substitution may be a conservative substitution. The collagens or polypeptides of the present invention may be derived from a peptide segment derived from type II human collagen. The collagens or polypeptides of the present invention may comprise a plurality of peptide segments thus obtained. The peptide segments can be linked via a linker or directly to form the collagens or polypeptides of the present invention. The linker may be one or more amino acid residues. For example, the linker may be a flexible linker commonly used in the art.

[0053] The present invention provides multiple repeat units, such as the underlined amino acid sequences in the Examples. In particular, the present invention provides a repeat unit of the amino acid sequence of SEQ ID NO. 15. The collagen or polypeptide of the present invention may contain multiple repeat units, for example, 2 to 30, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 repeat units. [Example]

[0054] The present invention is illustrated by the following examples. Those skilled in the art will understand that the examples are illustrative only and not limiting. The present invention is limited only by the appended claims.

[0055] Example 1 Construction and Expression of Recombinant Type II Humanized Collagen Large-scale screening of functional regions was carried out. First, sequence screening was carried out to eliminate regions that did not contain these important charge motifs, as the Gly-XY repeat sequence in the helical region of native type II collagen contains a large number of charged amino acids, and these charges interact to bind to cells. Second, computer-aided protein structure prediction was used to screen for potential helical functional regions with the most interchain hydrogen bonding structures and the most stable trimer aggregation. Third, protein expression prediction methods were used to screen for human type II collagen functional regions with the highest protein expression levels, ease of purification, and stability. Fourth, amino acid fragments from these regions were directly linked and optimized n times (the repetition was performed to ensure that the molecular weight of the recombinant humanized type II collagen was within a certain range, allowing for easy purification and stability), and the following target gene functional regions of different recombinant humanized type II collagen were obtained:

[0056] (1), the amino acid sequence of HC2B-a3: gkpgddgeagkpgkagergppgpqgargfgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagargndgqp (SEQ ID NO. 1), (2) Amino acid sequence of HC2B-a6: gkpgkagergppgpqgargfgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgppgppgpqgargfpgtpglpgvkghrgkpgkagppgppgpqgargfpgtpglpgvkghrgkpgkagppgppgpqgargfpgtpglpgvkghrgkpgkagergppgpgppgpqgargfgtpglpgvkghrgkpgkagergppgpgvkghrgkpgkagerg (SEQ ID NO. 2, the underlined portion indicates the repeating unit portion), (3), the amino acid sequence of HC2B-b1: gepgrespgadgppgrdgaagvkgdrgetgavgappgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpagpgpggprgppgvgpsgkdgangipgpigppgprgrsgetgpa (SEQ ID NO. 3), (4), HC2B-b3 amino acid sequence: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO. 4), (5), the amino acid sequence of HC2B-b5: gargiqgpqgprgdkgeagepgerglkghhrgftglqglpgppgps gargiqgpqgprgdkgeagepghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgprgdkgeagepgepgppgpglkghrgftglqgpgppgpsgargiqgpgpgrgftglqgpgpglkghrgftglqgpgppgpsglkghrgftglqglqgppgppgpsgargiqgpgpgqgpgdkgeagopgepglkghrgftglqgpgpgppgppgppgpsgargiqgpglkghrgftglqgppgppgps (SEQ ID NO. 5, the underlined parts indicate the repeating unit portions), (6), the amino acid sequence of B3Q: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerGlkghhrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO. 6, the underlined portion indicates the repeating unit portion), (7), the amino acid sequence of HC2B-A7: gpqgargfpgtpglpgvkghrgypgldgak gpqgargfgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgarggfgtpglpgvkghrgypgldgakgpqgfgtpglpgvkgghrgypgldgakgpqgarggtpglpgvkgghrgypgldgakgpqgarggfgtpglpglpgvkghrgypgldgakgldgakgpqgfggtpglpgvgghrgypgldgakgldgrgpgldgak (SEQ ID NO. 7, the underlined portion is the repeating unit portion), (8), the amino acid sequence of HC2B-A8: gargfpgtpglpgvkghrgypgldgak gargfgtpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgarggfpgtpglpgvkggggghrgfgggtpgvkghrgypgldgakgfpgglpglpgvkghrggpgldgakgargfpgtpgpglpgldgakgarggfpgpglpglpgvkggghrgypdgak (SEQ ID NO. 8, the underlined portion is the repeating unit portion), (9), the amino acid sequence of HC2B-A9: gfpgtpglpgvkghrgypgldgakgfpgtpglpgVkghrgypgldgakgfpggtpglpgvkghrgypgldgakgfpgtpgvkghrgypgldgakgfpggvgghrgypgldgakgfgtpglpgvkgghrgypgldgakgfgtpgvkghrgrgypgldgakgfgtpgvkghrgypgldgakgfgtpgvkghrgypgldgakgfgpgvgkghrgypgldgakgfgpgvgvkghrgpgldgakgfpglpgvgvgvkghrgpgpgldgakgpgldgakgfgpglpgvgvkgakgfgtpgpgvkghypgldgak (SEQ ID NO. 9, the underlined portion is the repeating unit portion), (10), amino acid sequence of HC2B-A10: gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak(SEQ ID NO.10), (11), the amino acid sequence of HC2B-B7: gerglkghhrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprggpglkghrgftglqgpgpgppgpsgpsgdqgasgpgpgpsgpgpsgpgpsgftglqgpgpgpgpglkghrgftglqgpgpgpgpgpsgdqgasgpgpgpglkghrgftglqgpgpgppgpsgdqgasgpgpgpglkgftglqgpgpgpgpgpsgdqgasgpgpgpgpgpsgdqgasgpgpgpgpgpsgpr (SEQ ID NO. 11, the underlined portion is the repeating unit portion), (12), the amino acid sequence of HC2B-B8: gerglkghhrgftglqglpgppgpsgdqgasgpa gerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghhrgftglqglp gppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghhrgftglqglpgppgpsgdqgasgpagerglkghhrgftglqglpgppgpsgdqgasgpa(SEQ ID NO.12), (13), the amino acid sequence of HC2B-B9: gerglkghrgftglqglpgppgpsgdq gerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgppgpsgdqgerglkghrgftglqglpgpggggpsgdqgerglkgghrgftglqglpggppgpsgdqgerglkrgftgl qglpggggpsgfglqgpggpgpsgdqgerglkghhrgftglqglqgpgpgpgpsgdqggghqgppgpgppgppgpgdq gppgppgppgppgpsgdqgppgppgppggpsgdqtglqgpgglkggggpsgdqftglqglqgpgpgpgpgsgdq(SEQ ID NO.13), (14), the amino acid sequence of HC2B-B10: gerglkghrgftglqglpgpp gerglkghrgftglqglpgppgerglkghhrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghhrgft glqglpgppgerglkghhrgftglqglpgppgerglkghhrgftglqglpgppgerglkghrgftglqglpgppgerglkghhrgftglqglpgppgerglkghhrgftglqglpgppgerglkghhrgftglqglpgppgerglkghhrgftglqglpgpp(SEQ ID NO.14).

[0057] The synthesized gene functional region is inserted into the pET-28a-Trx-His expression vector (see Figure 9) to obtain the corresponding recombinant expression plasmid.

[0058] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process is as follows: (1) E. coli competent cells BL21(DE3) were removed from the ultra-low temperature refrigerator and placed on ice. When partially thawed, 2 μl of the target plasmid was added to the E. coli competent cells BL21(DE3) and mixed thoroughly 2-3 times. (2) The mixture was then placed on ice for 30 min, heat-shocked in a 42°C water bath for 45-90 s, removed, and placed on ice for 2 min. (3) The cells were transferred to a biological safety cabinet, and 700 μl of liquid LB medium was added. The cells were then cultured at 37°C and 220 rpm for 60 min. (4) 200 μl of the bacterial suspension was evenly spread on an LB plate containing kanamycin sulfate (50 mg / L). (5) The plates were incubated in a 37°C incubator for 15 to 17 hours until uniformly sized colonies grew.

[0059] Five to six single colonies were selected from the transformed LB plate and placed in a shake flask containing antibiotic stock solution (ampicillin 100 mg / L). The resulting mixture was cultured at 220 rpm and 37°C in a constant temperature shaker until the mixture became mist-like. After culturing, the shake flask was cooled to 16-30°C, and IPTG (0.5 mM) was added to induce expression. The bacterial solution was then dispensed into centrifuge flasks and centrifuged at 6000 rpm for 12 minutes at 4°C. The bacterial cells were collected, their weights recorded, and samples were subjected to electrophoresis.

[0060] The collected cells were resuspended in an equilibrium working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), cooled to ≤15°C, and homogenized or ultrasonically disrupted twice. After completion, the cell suspension was collected. The disrupted cell suspension was dispensed into centrifuge flasks and centrifuged at 17,000 rpm at 4°C for 30 minutes, and the supernatant was collected.

[0061] Recombinant humanized type II collagen was purified and enzymatically cleaved. The specific process was as follows: (1) For crude purification, a) column equilibration was performed using an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) at a flow rate of 10 mL / min. b) For sampling, the supernatant after centrifugation was added to the column until the liquid stopped flowing, at a flow rate of 5 mL / min. c) For washing the hybrid protein, 100 mL of scrubbing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added until the liquid stopped flowing, at a flow rate of 10 mL / min. (d) To collect the target protein, 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) was added, and the flow rate was adjusted to 10 mL / min. The flow-through solution was collected and the protein concentration was measured by UV-visible spectrophotometry. The protein concentration was calculated using the following formula: C (mg / mL) = A280 × dilution factor × extinction coefficient. Then, electrophoretic detection was performed. (e) The column was washed with 1 M imidazole working solution, and the flow rate was adjusted to 10 mL / min. (2) For enzymatic cleavage, TEV enzyme was added so that the ratio of total protein to total TEV enzyme was 20:1. Enzymatic cleavage was performed for 2 hours at 16°C. The cleaved protein solution was placed in a dialysis bag and dialyzed at 4°C for 2 hours. It was then transferred to fresh dialysis solution (20 mM sodium chloride, 20 mM Tris) and dialyzed overnight at 4°C. (3) Regarding precision purification, a) for column equilibration, the column was equilibrated using Solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 ml / min. b) for sampling, the flow rate was set to 5 ml / min, and the flow-through sample was collected and electrophoretically detected. The protein was then stored at 4°C. c) for elution, the column was washed with 5 CV of Solution B (1 M sodium chloride, 20 mM Tris). d) The column was washed.

[0062] Regarding the test results, Figures 1 to 7 show the electrophoretic analysis results for each recombinant protein production process. Figure 1 shows the electrophoretic analysis results for recombinant type II humanized collagens HC2B-a6 and HC2B-b5, demonstrating low target protein expression after crude purification. Figure 2 shows the electrophoretic analysis results for recombinant type II humanized collagens HC2B-a3, HC2B-b1, and HC2B-b3, demonstrating the high level of hybrid protein after crude and refined protein purification. Figure 3 shows the electrophoretic analysis results for recombinant type II humanized collagens HC2B-A7 and HC2B-A8, demonstrating low target protein expression after crude purification. Figure 3 also shows the electrophoretic analysis results for recombinant type II humanized collagen B3Q, demonstrating low protein expression after refined protein purification. Figure 4 shows the electrophoretic analysis results for recombinant type II humanized collagen HC2B-A10, demonstrating high protein expression after crude purification, high enzymatic cleavage efficiency, and high protein purity after refined protein purification (left and center panels of Figure 4). Figures 4 and 7 further show the electrophoretic analysis results for recombinant type II humanized collagen HC2B-B9, indicating low crude protein expression, numerous extraneous bands, and poor enzymatic cleavage efficiency. Figure 5 shows the electrophoretic analysis results for recombinant type II humanized collagen HC2B-A9, indicating high crude protein expression, numerous impurity bands, and low purity. The left panel of Figure 6 shows the electrophoretic analysis results for recombinant type II humanized collagen HC2B-B8, indicating low crude protein expression, numerous impurity bands, and the absence of subsequent refinement. The right panel of Figure 6 and Figure 7 show the electrophoretic analysis results for recombinant type II humanized collagen HC2B-B7, indicating low crude protein expression, numerous impurity bands, and poor enzymatic cleavage efficiency. The center panel of Figure 6 shows the electrophoretic analysis results for recombinant type II humanized collagen HC2B-B10, indicating low crude protein expression, numerous impurity bands, and the absence of subsequent refinement.

[0063] Therefore, the recombinant type II humanized collagen HC2B-A10 achieves optimal efficiency in expression, isolation and purification compared with other recombinant type II humanized collagens.

[0064] Example 2: Detection of the biological activity of recombinant type II humanized collagen HC2B-A10 For a method of detecting collagen activity, see Juming Yao, Satoshi Yanagisawa, and 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). Specific implementation methods are as follows.

[0065] The concentrations of target protein samples, including a bovine type I collagen standard (Sigma, product code: 380002) and the recombinant type II humanized collagen HC2B-A10 (product code: 015) provided by the present invention, which has high purification and enzyme cleavage efficiency, were determined using the ultraviolet absorption method.

[0066] Specifically, the UV absorbance of the sample at 215 nm and 225 nm was measured, and the protein concentration was calculated using the empirical formula C (μg / mL) = 144 × (A215 - A225), with the caveat that detection is required when A215 is less than 1.5. The principle of this method is as follows: It measures the characteristic absorption of peptide bonds under far-UV light, is not affected by chromophore content, has few interfering substances, is easy to operate, and is suitable for measuring human collagen and its analogs, which do not react with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition, Humana Press, pp. 43-45.) After protein concentration detection, the concentration of all target proteins was adjusted to 0.5 mg / mL with PBS.

[0067] 100 μL of each protein solution and a blank PBS solution control were added to a 96-well plate and allowed to stand at room temperature for 60 minutes.

[0068] Each well contains 10 wells of good culture conditions. 5 3T3 cells were added and incubated at 37°C for 60 minutes.

[0069] Each well was washed four times with PBS.

[0070] The absorbance at OD492nm was detected using an LDH detection kit (Roche, 04744926001). The cell adhesion rate can be calculated based on the blank control value. The calculation formula is as follows:

number

[0071] As a result, as shown in Figure 8, the recombinant type II humanized collagen of the present invention has superior biological adhesive activity compared to bovine type I collagen (B col I), i.e., recombinant type II humanized collagen HC2B-A10 > bovine type I collagen. Unexpectedly, the inventors demonstrated that when the repeat sequence of HC2B-A10 is 8, the recombinant type II humanized collagen has a molecular weight that can be easily purified and stabilized, ensuring excellent biological adhesive activity.

[0072] Example 3 Mass Spectrometric Detection of Recombinant Type II Humanized Collagen HC2B-A10 Experimental Method [Table 1] The recombinant type II humanized collagen HC2B-A10 provided by the present invention, which is highly purified and has been enzymatically cleaved, was subjected to mass spectrometry detection, and the specific process is as follows:

[0073] Protein samples were reduced with DTT, alkylated with iodoacetamide, and then enzymatically digested overnight with trypsin. The resulting peptide fragments were desalted using a C18 ZipTip, mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA), and plated. Finally, the samples were analyzed using a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF / TOF) Ulraflextreme mass spectrometer. TM , Brucker, Germany (for peptide mass fingerprinting techniques see Protein J. 2016;35:212-7).

[0074] Data searches are performed from the MS / MS Ion Search page on the local masco site. Protein identification results are obtained from the primary mass spectra of peptide fragments generated after enzymatic digestion. For detection parameters, two uncleaved sites are set for trypsin enzymatic digestion. Cysteine ​​alkylation is set as a fixed modification. Methionine oxidation is set as a variable modification. The database used for identification is NCBprot.

[0075] Table 1. HC2B-A10 mass spectrometry detected molecular weight and corresponding polypeptide [Table 2] A coverage of the functional regions of the polypeptide was detected to be 100%.

[0076] GTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAK GTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAK (SEQ ID NO:30) DNA sequence of HC2B-a3 (SEQ ID NO.16): GGTAAACCAGGAGATGGAGAAGCAGGAAAACCAGGAAAAGCAGGAGAAAGAGACCGCCTGGACCGCAAGGAGCACGTGGATTTCCAGGAACCCCGGGACTGCCGGGTGTGAAAGGTCATAGAGGATATCCGGGACTGGATGGAGCAAAAGGAGAAGCAGGGGCACCGGGGAGTTAAAGGTGAGAGCGGAAGCCCGGGAAAAATGAAGCCCTGGTCCGATGGGTCCGAGAGGCTGCCGGGTGAAAGAGGGCGTACCGGTCGGCAGGAGCAGCAGGTGCAAGAGGAAATGATGGACAGCCG、 HC2B-α6 DNA sequence (SEQ ID NO.17): 、 DNA sequence of HC2B-b1 (SEQ ID NO.18): GTGAACCAGGTCGTGAAGGTAGCCCAGGTGCAGATGGACCACCAGGTCGTGATGGTGCAGCAGGAGTGAAAGGAGATCGTGGTGAAACCGGTGCAGTAGGTGCACCTGGTGCGCCAGGTCCGCCAGGTAGCCCTGGTCCAGCGGTCCTACCGGAAAAACAAGGGGATAGAGGGAGAAGCAGGAGCACAGGGTCCGATGGGTCCGTCAGGTCCGGCGGGTGCACGTGGTATTCAGGTCCGCAGGGTCCGCGTG GTGATAAAGGTGAAGCAGGTGAACCGGGGGAAAGAGGATTAAAAAGGGCATCGTGGTTTTACGGGTCTGCAGGTCTGCCTGGTCCGCCTGGTCGAGCGGTGATCAGGGTGCAAGCGGTCCGGCAGGTCCGAGCGGACCTCGTGGACCTCCGGGTCCTGTGGGTCCTAGTGGTAAGGATGGGGCAAATGGTATTCCTGGTCCTATTGGTCCCGGGTCCGCGTGGGAGATCAGGTGAACCGGACCGGCA、 DNA sequence of HC2B-b3 (SEQ ID NO.19): GGTAGCCCAGGTCCAGCAGGTCCGACAGGTAAACAAGGAGATCGTGGTGAAGCAGGAGCACAAGGACCAATGGGTCCAAGCGGTCCGGCAGGTGCAAGAGGTATTCAAGGGCCGCAAGGGCCACGGGTGATAAAGGGGAAGCAGGTGAACCAGGTGAGAGAGGGTTAAAAGGACATCGTGGATTTACAGGACTGCAGGTTTACCAGGTCCGCCGGGACCGAGCGGAGATCAAGGTGCAAGCGGTCCGGCGGGTCCGAGTCCTCGT、 DNA sequence of HC2B-b5 (SEQ ID NO.20): DNA sequence of B3Q (SEQ ID NO. 21): DNA sequence of HC2B-A7 (SEQ ID NO.22): 、 DNA sequence of HC2B-A8 (SEQ ID NO.23): GGTGCTAGGGGATTCCCGGGAACCCCGGGTCTGCCAGGCGTGAAAGGTCACCGCGGTTACCCGGGCCTCGACGGCGCGAAGGGTGCCCGTGGTTTTCCGGGAACCCCGGGCTTGCCAGGTGTCAAGGGCCATCGTGGTTACCCGGGTCTCGATGGTGCAAAGGGTGCGAGAGGCTTCCCGGGCACCCCGGGCCTGCCAGGGGTGAAAGGCCACAGAGGCTATCCTGGCTTGGATGGTGCCAAGGGTGCACGTGGATTCCCGGGCACTCCGGGTCTGCCGGGCGTGAAGGGCCACCGCGGTTATCCGGGCCTGGACGGTGCTAAAGGCGCGCGTGGTTTTCCGGGTACGCCGGGCTTGCCAGGTGTTAAGGGCCACCGTGGCTACCCGGGGCTGGATGGTGCCAAAGGTGCTCGCGGTTTCCCGGGAACCCCGGGTCTGCCTGGCGTGAAGGGTCATCGTGGTTACCCGGGCTTGGACGGCGCTAAGGGTGCGCGTGGTTTTCCGGGCACCCCGGGTCTGCCGGGGGTGAAAGGTCACCGCGGTTATCCCGGTCTGGATGGTGCGAAGGGTGCGCGTGGCTTCCCGGGCACCCCGGGCCTGCCGGGTGTTAAAGGTCATCGTGGTTACCCGGGCCTGGATGGTGCCAAGGGCGCTCGCGGTTTTCCGGGCACGCCAGGTTTACCGGGGGTCAAAGGCCATCGTGGCTATCCGGGTTTAGATGGCGCGAAAGGCGCACGCGGATTCCCGGGAACCCCGGGCCTGCCTGGCGTTAAAGGCCACCGCGGTTACCCGGGCCTTGACGGCGCGAAAGGCGCGCGTGGTTTTCCGGGCACCCCGGGTCTGCCGGGTGTTAAAGGTCACCGTGGCTATCCGGGTCTGGACGGTGCAAAAGGTGCACGTGG、 TTTCCCGGGGACTCCGGGCCTGCCGGGTGTTAAGGGCCATCGTGGTTACCCGGGTTTGGACGGTGCGAAG DNA sequence of HC2B-A9 (SEQ ID NO. 24): DNA sequence of HC2B-A10 (SEQ ID NO. 25): DNA sequence of HC2B-B7 (SEQ ID NO.26): GGAGAAAGGGGGTTGAAGGGACACCGCGGTTTTACTGGTTTGCAAGGCCTGCCGGGCCCTCCGGGTCCGTCTGGCGATCAGGGTGCAAGCGGCCCGGCGGGTCCGTCGGGCCCGCGTGGTGAGCGCGGTCTTAAGGGCCATCGTGGTTTCACCGGTTTACAAGGTCTGCCGGGCCCGCCGGGTCCGAGCGGTGATCAAGGGGCCTCCGGTCCGGCTGGCCCGTCCGGCCCAAGAGGCGAACGTGGTCTGAAAGGTCATCGTGGATTCACCGGACTGCAGGGTCTGCCTGGTCCGCCTGGTCCGTCAGGCGACCAAGGTGCGAGCGGTCCGGCGGGTCCGTCCGGTCCGCGTGGTGAACGTGGTCTGAAAGGCCACCGCGGCTTCACCGGTTTGCAAGGCCTGCCAGGCCCACCGGGTCCGTCTGGCGACCAGGGAGCCAGCGGTCCGGCTGGCCCATCTGGCCCACGCGGCGAGCGCGGTCTGAAAGGCCACCGTGGCTTTACGGGCTTGCAGGGTCTCCCGGGCCCACCGGGCCCGAGCGGTGATCAGGGTGCCAGCGGACCGGCAGGCCCCTCTGGTCCGCGTGGTGAACGTGGCCTGAAAGGTCATCGTGGTTTTACCGGTTTACAGGGCCTGCCAGGTCCCCCGGGTCCGTCCGGCGACCAGGGCGCAAGCGGTCCGGCTGGCCCGAGCGGTCCGCGTGGCGAGCGCGGCCTTAAGGGCCACAGAGGCTTCACGGGTCTGCAAGGTTTGCCGGGTCCGCCTGGCCCGTCGGGCGATCAGGGCGCGAGCGGCCCGGCGGGTCCGAGCGGTCCGCGTGGCGAGCGTGGTCTGAAGGGTCACCGCGGTTTTACCGGTCTGCAAGGTCTGCCGGGTCCGCCTGGCCCGAGCGGCGACCAGGGAGCGAGCGGTCCGGCGGGTCCGAGTGGTCCGCGT、 DNA sequence of HC2B-B8 (SEQ ID NO.27): GGGGAAAGGGGACTCAAAGGTCACCGCGGTTTCACGGGCCTTCAAGGTCTCGGGGTCCTCCGGGTCCGAGCGGCGACCAAGGTGCGTCTGGCCCAGCGGGTGAGCGTGGTTTAAAAGGCCACCGCGGTTTCACCGGCCTGCAGGGTTTACCGGGTCCGCCTGGCCGAGCGGTGATCAAGGTGCAAGCGGCCGGCAGGCGAAGCGGTCTG、 AAAGGCCATAGAGGTTTTACCGGCCTGCAGGGCTTGCCGGGCCCGCCGGGCCCGAGTGGCGATCAAGGTGCTTCCGGCCCGGCGGGTGAACGTGGCCTGAAGGGCCATCGTGGCTTTACCGGTCTGCAGGGCCTGCCAGGTCCGCCGGGCCCGTCTGGCACCAGGGTGCGA GCGGTCCAGCCGGTGAGCGCGGCTTGAAGGGCCACCGCGGCTTTACGGGTTTTGCAAGGTCTGCCTGGTCCGCCGGCCCGTCAGGCGATCAAGGGGCGAGCGGCCGCGGGTGAACGTGGTCTGAAAGGTCATCGTGGATTCACCGGCCTGCAGGGTCTGCCGGGCCCG、 CCGGCCCGTCCGGTGACCAGGGTGCTTCCGGTCCGGCTGGTGAGCGCGGTTTAAGGGACACCGTGGCTTCACCGGTCTGCAGGGCTTGCCGGGTCCTCCGGGCCGAGCGGCGACCAGGGTGCGTCCGGCCCGGCGGGTGAACGTGGTCTGAAGGGCCACCGTGGTTTTACCGCTTGCAAGGTCTGCCGGGTCCACCGGTCCGTCTGGCGATCA GGGAGCCAGCGGTCCGGCAGGCGAGCGTGGTTTGAAGGGTCACCGTGGATTCACCGGCCTGCAGGGCTGCCGGGCCCGCCGGGTCCGTCGGGCGATCAGGGAGCGAGCGGTCCCGGCAGGTGAGCGTGGTCTGAAAGGCCATCGTGGCTTCACTGGTTTGCAAGGCCTGCCTGGCCACCGGGTCCGAGCGGTGACCAGGGTGCCAGCGGTCGGCT、 DNA sequence of HC2B-B9 (SEQ ID NO.28): GGAGAAAGGGGGCTTAAGGGCCATCGTGGCTTCACGGGTTTGCAGGGCCTGCCGGGCCCACCGGGTCCGAGCGGCGATCAAGGCGAGCGGGGCCTGAAAGGTCATCGTGGCTTTACCGGTTTGCAGGGCCTGCCGGGCCCACCGGGACCGAGCGGTGATCAGGGGGAGCGTGGTCTGAAAGGTCATCGTGGTTTTACCGGTCTGCAGGGTTTGCCGGGTCCTCCGGGTCCGTCTGGCGACCAAGGTGAGCGTGGTCTGAAAGGTCATCGTGGTTTCACCGGTTTACAAGGCCTGCCGGGCCCACCGGGTCCGAGCGGTGACCAAGGCGAGCGTGGCCTGAAAGGCCACCGCGGTTTCACCGGTCTTCAGGGTCTGCCGGGCCCACCGGGTCCCAGCGGTGACCAGGGTGAACGCGGTCTGAAGGGTCACAGAGGCTTCACCGGTCTCCAAGGCTTACCGGGTCCGCCGGGCCCGAGCGGTGATCAGGGCGAACGTGGTCTGAAGGGCCACCGTGGCTTTACGGGTCTGCAAGGCCTGCCGGGTCCGCCGGGTCCGTCGGGCGACCAGGGTGAGCGCGGTTTGAAGGGCCACCGTGGATTCACCGGCCTGCAGGGCCTGCCGGGACCGCCTGGTCCGTCAGGTGATCAGGGTGAGCGCGGTCTCAAGGGTCATCGTGGTTTTACCGGTTTACAAGGTTTGCCGGGTCCGCCTGGCCCGTCTGGTGATCAAGGCGAACGTGGTCTGAAAGGCCACCGCGGCTTCACTGGTCTGCAGGGCTTGCCGGGCCCACCGGGTCCGTCCGGCGATCAAGGCGAACGTGGTCTGAAGGGTCACCGCGGATTCACGGGCCTGCAGGGCCTGCCGGGTCCGCCAGGTCCGTCCGGCGACCAGGGCGAACGCGGGCTGAAAGGTCACCGTGGCTTTACCGGTTTGCAAGGACTGCCGGGTCCGCCGGGCCCGAGCGGCGACCAG、 DNA sequence of HC2B-B10 (SEQ ID NO. 29):

Claims

1. A collagen comprising a plurality of repeat units comprising the amino acid sequence set forth in SEQ ID NO. 15, an amino acid sequence obtained by substituting, inserting, deleting, or adding one to four amino acid residues in the amino acid sequence of SEQ ID NO. 15, or an amino acid sequence having 90% to 99% identity to the amino acid sequence of SEQ ID NO. 15, wherein each repeat unit is directly linked and the number of repeat units is two or more, the collagen is recombinant type II humanized collagen, and has cell adhesion activity, and optionally the number of repeat units is 6 to 10.

2. 2. The collagen of claim 1, wherein the collagen comprises the amino acid sequence shown in SEQ ID NO. 10, an amino acid sequence obtained after the amino acid sequence of SEQ ID NO. 10 has undergone mutations such as substitution, insertion, deletion, or addition of 1 to 32 amino acid residues, or a variant having 90% to 99% identity with the amino acid sequence of SEQ ID NO.

10.

3. 10. A polynucleotide encoding the collagen of claim 1, optionally comprising the nucleotide sequence set forth in SEQ ID NO.

29.

4. 4. A nucleic acid comprising the polynucleotide of claim 3, optionally further comprising nucleotides encoding a purification tag, optionally a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, and optionally further comprising nucleotides encoding a leader sequence.

5. 4. A vector comprising the polynucleotide of claim 3, optionally an expression vector, and optionally comprising expression control elements, optionally a promoter, terminator and / or enhancer, operably linked to said polynucleotide.

6. 4. A host cell comprising the polynucleotide of claim 3, and optionally being a bacterial, fungal or animal cell, optionally wherein the bacterium is Escherichia coli, and optionally wherein the fungus is a yeast, optionally Saccharomyces cerevisiae.

7. (1) culturing host cells under suitable culture conditions; (2) harvesting the host cells and / or medium containing collagen; 3. A method for producing collagen according to claim 1 or 2, comprising the step (3) of purifying the collagen.

8. A composition comprising the collagen of claim 1 or 2, and optionally one or more of a bio-covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a 3D printing artificial organ biomaterial, a cosmetic raw material, a medicinal supplement, and a food additive, and optionally an injectable composition or an oral composition.

9. 9. The composition of claim 8, which is a composition for cartilage repair, optionally an injectable composition for cartilage repair.

10. 10. An in vitro method for enhancing cell adhesion, comprising contacting cells with the collagen of claim 1 or 2.

11. A kit for enhancing cell adhesion or cartilage repair, or a method for producing one or more of a biological covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a medicinal auxiliary material and a food additive, the method comprising a step of using the collagen described in claim 1 or 2, the polynucleotide described in claim 3, the nucleic acid described in claim 4, the vector described in claim 5, and / or the host cell described in claim 6.

12. A kit comprising the collagen described in claim 1 or 2.

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