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 the priority of a Chinese patent application with an application date of May 12, 2023, an application number of 202310537499.9, and an invention title of "Polypeptide and Its Use". The above application content is incorporated herein by reference.

[0002] The present invention belongs to the technical field of synthetic biology, and specifically relates to human body structural materials and their biosynthetic manufacturing methods.

Background Art

[0003] Collagen is a type of protein widely distributed in the connective tissues of the human body. It is also the most abundant protein in the human body, accounting for 25% - 35% of the total protein content. Currently, there are at least 28 collagen subtypes in the human body, and they have been found to be located in different tissues and organs respectively.

[0004] Type II collagen is mainly present in cartilage tissue, vitreous body and cornea. It is a kind of macromolecular protein. Its filamentous collagen fibers are intertwined with elastin and polysaccharide proteins to form a network structure, and it is also called composite collagen. Since type II collagen is a necessary component for cartilage and bone morphogenesis, bone growth and maintenance of mature cartilage, non-denatured collagen can be a structural and functional component of cartilage.

[0005] Type II collagen, as a major component of articular cartilage matrix, protects cartilage from wear together with lubricating components such as hyaluronic acid and proteoglycans. With aging, the synthesis rate of collagen gradually decreases, the loss of collagen becomes severe, articular cartilage undergoes degenerative changes, the friction between bones intensifies, and inflammation is caused in the joints. Currently, the common treatment method for osteoarthritis is to consume collagen, but the utilization rate of this protein is low, the regeneration rate of cartilage is slow, which is disadvantageous for the rehabilitation of patients and affects the treatment process and efficiency. Since the current treatment means do not have a favorable treatment effect, patients ultimately have to choose artificial joint replacement, which is not only costly but also has the possibility of fatal complications such as thrombosis formation after the operation and limb amputation due to infection after the replacement.

[0006] However, the collagen materials currently used as cartilage mainly originate from the extraction of animal cartilage. The immunogenicity of animal-derived collagen cannot be removed, and its impurity removal and extraction processes are still complex. It is difficult to guarantee the triple helix structure of collagen and it cannot be used for large-scale production.

[0007] At the same time, the immune reaction derived from animals is also an important cause restricting the application of collagen. With the increasing expansion of the collagen industry in China, obtaining collagen using biosynthetic pathways is becoming increasingly mature, especially humanized collagen leading the world. In 2021, the China National Medical Products Administration carried out naming and classification for biosynthetic collagen. Recombinant humanized collagen refers to the functional region of the full-length or partial amino acid sequence encoded by a specific human collagen gene produced by DNA recombination technology, or a combination of functional regions containing human collagen function.

[0008] Conventional methods for producing collagen involve treating animal-derived tissues using acid, alkali, and enzymatic degradation methods to extract collagen derivatives. The collagen extracted by these methods has lost its original biological activity and cannot actually function when applied in the biomedical field. With the development of modern technology, several extraction methods for obtaining non-denatured type II collagen by removing impurities and performing enzymatic degradation extraction on animal cartilage have emerged both at home and abroad. The produced collagen can be applied to cartilage repair, but this method has a long time for impurity removal and extraction, low product purity, low product stability, and is disadvantageous for mass production. In response to the deficiencies of the prior art, a company proposed to produce type II collagen for cartilage repair using Pichia yeast as the host strain. However, since this collagen does not belong to humanized collagen, it has a certain immunogenic reaction.

[0009] Therefore, in order to be applied to cartilage repair as a human body structural material, there is a strong demand for recombinant type II humanized collagen that can be directly injected into the human body and does not cause an immunogenic reaction.

Summary of the Invention

Problems to be Solved by the Invention

[0010] Human structural materials mainly include structural proteins in collagen. Such proteins have complex structures, accurate functions, and are of great significance for the repair and regeneration of human tissues, but they cannot be easily obtained by ordinary production methods. The present invention develops a polymer functional protein with a triple helix structure of human type II collagen and capable of functioning as human collagen by means of synthetic biology and structural biology technologies. The inventors have conducted large-scale screening for functional regions. First, sequence screening is carried out. A large number of charged amino acids are contained in the Gly-X-Y repeated gene sequence in the helical region of natural type II collagen, and these charges bind to cells through interactions. Therefore, regions without these important charge motifs are excluded. Second, by means of a method for predicting protein structures assisted by a computer, it contributes to screening potential helical functional regions with the most inter-chain hydrogen bond structures and capable of most stabilizing the trimer aggregation form. Third, based on a method for predicting protein expression properties, a functional region of human type II collagen with the highest protein expression level, easy to purify, and high stability is screened. Fourth, the amino acid fragments of these regions are directly linked and optimized by repeating them n times (the repetition is carried out to ensure that the molecular weight of recombinant type II humanized collagen is within a certain range and can be easily purified and stabilized).

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

[0012] A part of the present invention is based on the following unexpected findings of the inventors. Compared with other recombinant type II humanized collagens, recombinant type II humanized collagen HC2B-A10 has a larger expression level during recombinant expression, is easier to separate and purify, and has a higher cell adhesion effect compared to bovine type I collagen.

Means for Solving the Problems

[0013] In one aspect, the collagen or polypeptide according to the present invention includes a plurality of repeating units including 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, and each repeating unit is directly linked or separated by one or more amino acid residues. In one embodiment, the repeating unit may be an amino acid sequence obtained after the amino acid sequence of SEQ ID NO. 15 has undergone a mutation (substitution, insertion, deletion, or addition) of one or more amino acid residues.

[0014] In one embodiment, the number of repeating units is from 1 to 20. In one embodiment, the number of repeating units is from 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 includes the amino acid sequence shown in SEQ ID NO. 10. In one embodiment, the collagen or polypeptide includes the amino acid sequence shown 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 a 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%, or 99% identity to the amino acid sequence shown in SEQ ID NO. 10. In one embodiment, when the collagen or polypeptide sequence mutates, the resulting collagen or polypeptide retains the functions of the present invention such as cell adhesion and cartilage repair ability.

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

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

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

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

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

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

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

[0023] In one aspect, the composition according to the present invention contains the collagen or polypeptide described herein. In one embodiment, the composition is a kit. In one embodiment, the composition is one or more of a biological coating material, a human biomimetic material, a plastic and aesthetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration material, a liver tissue material, a blood vessel repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a pharmaceutical adjuvant material, and a food additive, and preferably is 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, the method for enhancing cell adhesion according to the present invention includes the step of contacting cells with the collagen or polypeptide described herein, the polynucleotide described herein, the nucleic acid described herein, the vector described herein, the host cell described herein, and / or the composition described herein.

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

[0027] The advantages of the present invention include the following.

[0028] 1. The present invention provides the core functional region and amino acid sequence of recombinant type II humanized collagen. 2. The present invention has successfully synthesized recombinant type II humanized collagen that can be injected into the human body and is used for cartilage repair for the first time. 3. The produced recombinant type II humanized collagen has an amino acid composition that is 100% identical to the corresponding part of the natural collagen amino acid sequence and does not cause immune rejection and allergic reactions when applied to the human body. 4. The manufacturing method described herein is simple and can produce a high yield of recombinant type II humanized collagen on a large scale. 5. Recombinant type II humanized collagen HC2B-A10 achieves an optimal effect in terms of the effects of expression, separation, and purification compared to other recombinant type II humanized collagens. 6. The recombinant type II humanized collagen of the present invention has better biological adhesion activity, that is, recombinant type II humanized collagen HC2B-A10 > bovine type I collagen.

Brief Description of the Drawings

[0029]

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Modes for Carrying Out the Invention

[0030] To make the objectives, technical means and advantages of the present invention clearer, the following refers to the embodiments of the present invention to clearly and completely describe the technical means in the embodiments of the present invention. Obviously, the described embodiments are some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments that can be conceived by those skilled in the art without creative efforts all belong to the protection scope of the present invention.

[0031] Recombinant collagen utilizes technologies such as advanced structural biology and genetic engineering to screen and manufacture a novel biomaterial that is identical or similar to the human collagen amino acid sequence, using the gene code of the functional region of human-specific collagen as a template.

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

[0033] In this specification, collagen or polypeptide may contain one or more repeating units including the amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence obtained after the amino acid sequence has undergone mutations (substitutions, additions, insertions, or deletions) of one or more amino acid residues. The number of repeating units is from 1 to 20. For example, 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. In particular, the mutation may be a substitution, such as a conservative amino acid substitution. The amino acid sequence of SEQ ID NO.15 is gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak. Each repeating unit may be directly linked or separated by one or more amino acid residues.

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

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

[0036] As used herein, the term "vector" is a nucleic acid delivery tool into which a polynucleotide can be inserted. A vector is called an expression vector if it can express the protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, and express the genetic material element carried therein 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 λ phage or M13 phage, and animal viruses. Vectors can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can further contain an origin of replication. A vector can contain the nucleic acid of the present invention for introduction into a cell for expression. The vector can contain expression control elements, such as a promoter, a terminator, and / or an enhancer, operably linked to the nucleic acid.

[0037] As used herein, the term "host cell" is 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 into naked DNA by acceleration with electroporation, lipid transfection, and particle guns. The host cell may be a eukaryotic cell or a prokaryotic cell. For example, the eukaryotic cell may be a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell may be an E. coli cell.

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

[0039] The collagen or polypeptide of the present invention can be manufactured into a composition or a kit. The composition or kit may be a composition or kit for tissue filling and / or solubilization. The composition or kit may further contain auxiliary substances. The composition of the present invention may be a cartilage repair agent containing the collagen or polypeptide described herein. The composition of the present invention may be injectable. The composition of the present invention may be, for example, a human structural material that can be used for cartilage repair and does not cause 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 the purposes of the present invention, 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: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) is used to determine the sequence identity between two amino acid sequences. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of needle with the tag "longest identity" (obtained using the non-abbreviated option) is used as the percentage identity and calculated as follows.

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

[0042] (Identical deoxyribonucleotides × 100) / (Alignment length - Total number of gaps in the alignment) In this specification, the repeating units of the collagen or polypeptide of the present invention, or the collagen or polypeptide, may have certain mutations. For example, one or more amino acid sequences of these portions may have substitutions, deletions, additions, or insertions of amino acid residues. In the context of amino acid mutations, "plurality" may be 2 to 40, for example, 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 numerical range therebetween.

[0043] That is, the present invention can use repeating unit mutants as long as they have the activity of promoting cell adhesion. Specifically, the mutant may have a certain percentage identity with a specific sequence (any collagen or polypeptide sequence described herein), for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The specific sequence may be any sequence of the present invention, such as SEQ ID NOs. 1 to 16, but these mutants preferably retain the intended functions such as enhancing cell adhesion or cartilage repair.

[0044] The method of the present invention In view of the current research status, the present invention provides a method for biosynthesizing recombinant type II humanized collagen, that is, a method for manufacturing a human structural material. The method may include one or more of the steps of screening functional regions and constructing bacterial strains (step (1)), large-scale bioreactor culture and induced expression of proteins (step (2)), and purification of humanized type II collagen and optional enzymatic cleavage (step (3)).

[0045] The screening of functional regions and the construction of bacterial strains can be carried out as follows. (1) Screen the functional regions on a large scale to obtain the target gene functional region. (2) Insert the obtained target gene functional region into the PET-28a-Trx-His expression vector to obtain a recombinant expression plasmid. (3) Introduce the recombinant expression plasmid into Escherichia coli competent cells BL21(DE3), and screen to obtain positive Escherichia coli genetic engineering bacteria.

[0046] Large-scale biological fermentation can be carried out as follows. The positive Escherichia coli genetic engineering bacteria obtained by screening were added to an antibiotic stock solution shake flask and cultured with a constant temperature shaker at 220 rpm and 37 °C.

[0047] The induced expression of the protein can be carried out as follows. (1) Cool the cultured shake flask to 16 - 30 °C. (2) Add the IPTG stock solution to perform induced expression. (3) Put the bacterial liquid after induced expression into a centrifuge flask, centrifuge at 6000 rpm and 4 °C for 12 min, and then collect the bacterial cells.

[0048] The purification and optional enzymatic cleavage of humanized type II collagen can be carried out as follows. (1) Crudely purify humanized type II collagen with a Ni affinity chromatography column. (2) Add TEV enzyme at a certain ratio for enzymatic cleavage. (3) Precisely purify humanized type II collagen with an ion exchange column.

[0049] The screened functional regions are shown below. (1) Amino acid sequence of HC2B-a3: gkpgddgeagkpgkagergppgpqgargfgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagargndgqp (SEQ ID NO:1), (2), Amino acid sequence of HC2B-a6: gkpgkagergppgpqgargfgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgkagergppgpqgargfgtpglpgvkghrgkpgkagergppgpgvkghrgkpgvkghrgkpgkagppgpqgargfpgtpglpgvkgrgkpgkpgkagerg (SEQ ID NO:2) , (3), Amino acid sequence of HC2B-b1: gepgregrespgadgppgrdgaagvkgdrgetgavgappgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpagpsgppgppgppgvgpsgkdgangipgpigppgprgrsgetgpa (SEQ ID NO:3) , (4), Amino acid sequence of HC2B-b3: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO:4) , (5), Amino acid sequence of HC2B-b5: gargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgpqgqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgqgprgdkgeagopgepgppgargiqgpgpglkghrgftglqglpgppgpglkghrgftglqgpgppgpglkghrgftglqgppgppgpgpsgargiqgpgqgpprgdkgeagopgepglkghrgftglqglpgppgppgppgppgpgppgpsgargiqgpgqgpgppgppgpgps (SEQ ID NO:5), (6), Amino acid sequence of B3Q: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpgqgprgdkgeagepgerglkgghrgftglqglpgppgpsgdqgasgpagpsgpsggpgpagptgkqgdrgagaqgrgargiqgpgqgrgdkgeagepgerglkggggghrgftglqglqglpgppgpsgpsgdqgasgpagpgptgkqgdrgagarggaqgpsgpstgkqgargiqgqgpqgpgqgprgqgprgdkgepgepglkghftglqgppgpsgpsgdqgqgqgas (SEQ ID NO:6) , (7), Amino acid sequence of HC2B - A7: gpqgargfpgtpglpgvkghrgypgldgakgpqgargfgtpglpgvkghrgypgldgakgpqgargfpggtpglpgvkghrgypgldgakgpqgargfgtpglpgvkghrgypgldgakgpqgarggfgtpglpgvkghrgypgldgakgpqgarggtpglpgvkghrgypgldgakgqgarggfgtpglpgvkghrgypgldgakgpqgldgakgldgakgpqgldgakgpgpglpgvkghrgypgldgak (SEQ ID NO:7) , (8), Amino acid sequence of HC2B - A8: gargfpgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkgghrgypgldgakgargfggtpglpgvkghrgypgldgakghrgfgggldgakghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgldgakgargfgtpglpglpgvkgggghrgdgakgldgak (SEQ ID NO:8), (9), Amino acid sequence of HC2B-A9: gfpgtpglpgvkghrgypgldgakgfpgtpglpgvkghrgypgldgakgfpglpgvkghrgypgldgakgldgakgfpgtpglpgvkghrgypgldgakgfpgvgghrgypgldgakgfgtpglpgvkghrgypgldgakgldgakgfgtpglpgvkghrgypgldgakgfgtpgvkghrgypgldgakgldgakgfpglpgvkghrgypgldgakgvkghrgypgldgakgldgakglpgvkghrggypgldgakgfglpgvkghrggggfgtpgvgrggdgakglpgldgakgpgldgakgldgak (SEQ ID NO:9), (10), Amino acid sequence of HC2B-A10: gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgakgtpgvkgghrgypgldgakgtpglpgvkghrgypgldgakgldgakgtpgvkgghrgypgldgakgldgakgtpgvkghrgypgldgakglpgvkghrgypgldgakgtpgvkghrgypgldgakgldgakgtpglpgvkghrggdgakgypgldgakgldgakgtpglpgvkghrggypgldgak (SEQ ID NO:10) , (11), Amino acid sequence of HC2B-B7: gerglkghrgftglqglpgppgpsgdqgasgpagpsgprgglkghrgftglqglpgppgpsgdqgasgpagpsgprggpglkghrgftglqglpgppgpsgpsgdqgasgpgpgpsgpsgdqgasgpgpglkghrgftglqglpgppgppgpsgdqgasgpgpgpgpsgrgftglqglpgpgpsgpgpsgpsgdqgasgpgpglkghrgftglqglqgpgpgpgppgpsgdqgasgpgpgpsgpr (SEQ ID NO:11), (12), Amino acid sequence of HC2B - B8: gerglkghrgftglqglpgppgpsgdqgasgpaglkghrgftglqglpgppgpsgdqgasgpaglkghrgftglqglpgpgpgpgdqgasgpglkghrgftglqglpgppgpsgdqgasgpaglkghrgftglqgpgpgpgpgpgdqgasglkghrgftglqgpgppgpgpgpgpgpggpgftglqgpgppgpgpgpsgdqgasgpglkghrgftglqglpgppgpgpsgdqgasgpgpgpgpgpgftglqglkghrgftglqgpggppgpgpgdqgasgpgpa (SEQ ID NO:12) , (13), Amino acid sequence of HC2B - B9: gerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglqglpgpgppgpsgdqgerglkghrgftglqglqglpgpgppgpsgdqglkghrgftglqglpggppgpsggdqgerglkrgftglqglpggggpsgfglqgppgpsgdqgerglkghrgftglqglqgpgpgpgpsgdqggghqgppgpgpgpsgdqgppgppgppgpsgdqglkgrgftglqglpggggpsgdq (SEQ ID NO:13) , (14), Amino acid sequence of HC2B - B10: gerglkghrgftglqglpgppgglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpggppgglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgppgerglkghrgftglqglkghrgftglqglpgppglkghrgftglqgppglkghrgftglqgpgppglkgftglqgpgppgppgppglkghrgftglqglkghrgftglqgpgpp (SEQ ID NO:14) .

[0050] The recombinant type II humanized collagen amino acid sequence produced by the present invention can be derived from the functional region of human native 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] Since the recombinant type II humanized collagen produced by the present invention completely matches the amino acid sequence of human collagen, it can be directly injected into the human body without causing an immunogenic reaction and used for cartilage repair.

[0052] The collagen or polypeptide of the present invention The present invention provides a collagen or polypeptide 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 collagen or polypeptide of the present invention may be derived from a peptide segment derived from type II human collagen. The collagen or polypeptide of the present invention may contain a plurality of peptide segments thus obtained. The peptide segments can form the collagen or polypeptide of the present invention by a linker or direct linkage. 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 a plurality of repeating units such as the amino acid sequences indicated by the underlines in the examples. In particular, the present invention provides a repeating unit of the amino acid sequence of SEQ ID NO. 15. The collagen or polypeptide of the present invention may contain a plurality of repeating 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.

Examples

[0054] The present invention will be described by the following examples. Those skilled in the art should understand that the examples are merely illustrative and not restrictive. The present invention is limited only by the appended patent claims.

[0055] Example 1 Construction and Expression of Recombinant Type II Humanized Collagen Perform large-scale screening on the functional regions. First, perform sequence screening. Since the Gly-X-Y repeat gene sequence in the helical region of natural type II collagen contains a large number of charged amino acids, and these charges bind to cells through interactions, exclude the regions that do not contain these important charge motifs. Second, contribute to screening potential helical functional regions that have the most inter-chain hydrogen bond structures and can most stably maintain the trimer aggregation form by the method of predicting protein structure with computer assistance. Third, based on the method of predicting protein expression properties, screen the human type II collagen functional regions with the highest protein expression level, easy to purify, and high stability. Fourth, repeat the amino acid fragments of these regions n times (the repetition is performed to ensure that the molecular weight of the recombinant type II humanized collagen is within a certain range and can be easily purified and stabilized), directly ligate and optimize them to obtain the following target gene functional regions of different recombinant type II humanized collagens.

[0056] (1) Amino acid sequence of HC2B-a3: gkpgddgeagkpgkagergppgpqgargfgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagargndgqp (SEQ ID NO.1), (2) Amino acid sequence of HC2B-a6: gkpgkagergppgpqgargfgtpglpgvkghrgkpgkagergppgpqgargfpgtpglpgvkghrgkpgppgppgpqgargfpgtpglpgvkghrgkpgkagppgppgpqgargfpgtpglpgvkghrgkpgkagergppgpgppgpqgargfgtpglpgvkghrgkpgkagergppgpgvkghrgkpgkagerg(SEQ ID NO.2, the underlined part is the repeating unit), (3) Amino acid sequence of HC2B-b1: gepgrespgadgppgrdgaagvkgdrgetgavgappgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpagpgpggprgppgvgpsgkdgangipgpigppgprgrsgetgpa (SEQ ID NO.3), (4) Amino acid sequence of HC2B-b3: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO.4), (5) Amino acid sequence of HC2B-b5: gargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgps gargiqgpqgprgdkgeagepghrgftglqglpgppgpsgargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgargiqgprgdkgeagepgepgppgpglkghrgftglqgpgppgpsgargiqgpgpgrgftglqgpgpglkghrgftglqgpgppgpsglkghrgftglqglqgppgppgpsgargiqgpgpgqgpgdkgeagopgepglkghrgftglqgpgpgppgppgpgpsgargiqgpglkghrgftglqgppgppgps (SEQ ID NO.5, the underlined part is the repeating unit), (6) Amino acid sequence of B3Q: gspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerGlkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgprgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgeagepgerglkghrgftglqglpgppgpsgdqgasgpagpsgpr (SEQ ID NO.6, the underlined part is the repeating unit), (7) Amino acid sequence of HC2B - A7: gpqgargfpgtpglpgvkghrgypgldgak gpqgargfgtpglpgvkghrgypgldgakgpqgargfpgtpglpgvkghrgypgldgakgpqgarggfgtpglpgvkghrgypgldgakgpqgfgtpglpgvkgghrgypgldgakgpqgarggtpglpgvkgghrgypgldgakgqgarggfgtpglpglpgvkghrgypgldgakgldgakgpqgfggtpglpgvgghrgypgldgakgldgrgpgldgak (SEQ ID NO.7, the underlined part is the repeating unit), (8) Amino acid sequence of HC2B - A8: gargfpgtpglpgvkghrgypgldgak gargfgtpgvkghrgypgldgakgargfpgtpglpgvkghrgypgldgakgargfpgtpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgargfgtpglpgvkghrgypgldgakgarggfpgtpglpgvkggggghrgfgggtpgvkghrgypgldgakgfpgglpglpgvkghrggpgldgakgargfpgtpggvkghrgggldgakgfpggtpgpglpgldgakgarggfpgpglpglpgvkggghrgypdgak (SEQ ID NO.8, the underlined part is the repeating unit), (9) Amino acid sequence of HC2B - A9: gfpgtpglpgvkghrgypgldgakgfpgtpglpgVkghrgypgldgakgfpggtpglpgvkghrgypgldgakgfpgtpgvkghrgypgldgakgfpggvgghrgypgldgakgfgtpglpgvkgghrgypgldgakgfgtpgvkghrgrgypgldgakgfgtpgvkghrgypgldgakgfgpgvgkghrgypgldgakgfgpgvgvkghrgpgldgakgfpglpgvgvgvkghrgpgpgldgakgpgldgakgfgpglpgvgvkgakgfgtpgpgvkghypgldgak (SEQ ID NO.9, the underlined part is the repeating unit), (10) Amino acid sequence of HC2B - A10: gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak Gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak gtpglpgvkghrgypgldgakgtpglpgvkghrgypgldgak (SEQ ID NO.10), (11) Amino acid sequence of HC2B - B7: gerglkghrgftglqglpgppgpsgdqgasgpagpsgprgerglkghrgftglqglpgppgpsgdqgasgpagpsgprggpglkghrgftglqgpgpgppgpsgpsgdqgasgpgpgpsgpgpsgftglqgpgpgpgpglkghrgftglqgpgpgpgpgpgpsgdqgasgpgpgpglkghrgftglqgpgpgppgpsgdqgasgpgpgpglkgftglqgpgpgpgpgpgpsgdqgasgpgpgpgpgpgpsgpr(SEQ ID NO.11, the underlined part is the repeating unit part), (12) Amino acid sequence of HC2B - B8: gerglkghrgftglqglpgppgpsgdqgasgpa gerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpagerglkghrgftglqglpgppgpsgdqgasgpa(SEQ ID NO.12), (13) Amino acid sequence of HC2B - B9: gerglkghrgftglqglpgppgpsgdq gerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgpsgdqgerglkghrgftglqglpgppgppgpsgdqgerglkghrgftglqglpgpggggpsgdqgerglkgghrgftglqglpggppgpsgdqgerglkrgftglqglpggggpsgfglqgpggpgpsgdqgerglkghrgftglqglqgpgpgpgpsgdqggghqgppgpgppgppgpgdqgppgppgppgppgpsgdqgppgppgppggpsgdqtglqgpgglkggggpsgdqftglqglqgpgpgpgpgsgdq(SEQ ID NO.13), (14) Amino acid sequence of HC2B-B10: gerglkghrgftglqglpgpp gerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgppgerglkghrgftglqglpgpp(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 Escherichia coli competent cells BL21(DE3). The specific process is as follows. (1) Take out Escherichia coli competent cells BL21(DE3) from an ultra-low temperature refrigerator and place them on ice. When they are semi-thawed, add 2 μl of the plasmid to be transformed to Escherichia coli competent cells BL21(DE3) and mix evenly about 2 - 3 times. (2) After incubating the mixture on ice for 30 min, perform heat shock in a 42°C water bath for 45 - 90 s. After taking it out, incubate it on ice for 2 min. (3) Transfer it to a biological safety cabinet, add 700 μl of liquid LB medium, and culture it at 37°C and 220 rpm for 60 min. (4) Spread 200 μl of the bacterial solution evenly on an LB plate containing kanamycin sulfate (50 mg / L). (5) Culture the plate in a 37°C incubator for 15 - 17 h until colonies of uniform size grow.

[0059] Select 5 to 6 single colonies from the transformed LB plates, place them in an Erlenmeyer flask containing an antibiotic stock solution (100 mg / L ampicillin), and culture them for a certain period of time on a constant-temperature shaker at 220 rpm and 37 °C until they become misty. Further, cool the cultured Erlenmeyer flask to 16 - 30 °C, add IPTG (0.5 mM), induce expression for a certain period of time, then dispense the bacterial solution into a centrifuge flask, centrifuge at 6000 rpm and 4 °C for 12 min, collect the bacterial cells, record the weight of the bacterial cells, sample, and perform electrophoresis detection.

[0060] Resuspend the collected bacterial cells in an equilibration buffer (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), cool the bacterial solution to ≤15 °C, homogenize twice or disrupt the cells by sonication, and after completion, collect the bacterial solution. Dispense the bacterial solution after cell disruption into a centrifuge flask, centrifuge at 17000 rpm and 4 °C for 30 min, and collect the supernatant.

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

[0062] Regarding the test results, The results of electrophoretic detection regarding the manufacturing process of each recombinant protein are shown in FIGS. 1 to 7. FIG. 1 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-a6 and HC2B-b5, indicating that the expression level of the target protein after crude purification is low. FIG. 2 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-a3, HC2B-b1, and HC2B-b3, indicating that there are many hybrid proteins after crude purification and precision purification of the protein. FIG. 3 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-A7 and HC2B-A8, indicating that the expression level of the target protein after crude purification is low. FIG. 3 further shows the electrophoretic detection results of recombinant type II humanized collagen B3Q, indicating that the protein expression level after precision purification is low. FIG. 4 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-A10, indicating that the protein expression level in crude purification is high, the enzyme cleavage effect is high, and the purity of the protein after precision purification is high (the left and central figures in FIG. 4). FIGS. 4 and 7 further show the electrophoretic detection results of recombinant type II humanized collagen HC2B-B9, indicating that the protein expression level in crude purification is low, there are many extra bands, and the enzyme cleavage effect is low. FIG. 5 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-A9, indicating that the protein expression level in crude purification is high, there are many impurity bands, and the purity is low. The left figure in FIG. 6 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-B8, indicating that the protein expression level in crude purification is low, there are many impurity bands, and subsequent precision purification is not performed. The right figure in FIG. 6 and FIG. 7 show the electrophoretic detection results of recombinant type II humanized collagen HC2B-B7, indicating that the protein expression level in crude purification is low, there are many impurity bands, and the enzyme cleavage effect is low. The central figure in FIG. 6 shows the electrophoretic detection results of recombinant type II humanized collagen HC2B-B10, indicating that the protein expression level in crude purification is low, there are many impurity bands, and subsequent precision purification is not performed.

[0063] Therefore, compared with other recombinant type II humanized collagens, recombinant type II humanized collagen HC2B-A10 achieves the optimal effect in terms of the effects of expression, separation, and purification.

[0064] Example 2 Detection of the biological activity of recombinant type II humanized collagen HC2B-A10 For the method of detecting the activity of collagen, reference can be made to 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.

[0065] Using the ultraviolet absorption method, the concentrations of bovine type I collagen standard product (Sigma, number: 380002) and the protein sample to be detected containing recombinant type II humanized collagen HC2B-A10 (number 015) with high purification and enzyme cleavage effects provided by the present invention were detected.

[0066] Specifically, the ultraviolet light absorption of the sample at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated by the empirical formula C (μg / mL) = 144×(A215 - A225). Note that it is necessary to detect when A215 < 1.5. The principle of this method is as follows. 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 easy to operate, and is suitable for the measurement of human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43~45). After detecting the protein concentration, the concentrations of all proteins to be measured were adjusted to 0.5 mg / mL with PBS.

[0067] 100 μL of various protein solutions and blank PBS solution control were added to a 96-well plate and left standing at room temperature for 60 min.

[0068] 10 3T3 cells with good culture status were added to each well and incubated at 37 °C for 60 min. 5 Each well was washed 4 times with PBS.

[0069] The absorbance at OD492 nm was detected using an LDH detection kit (Roche, 04744926001). Based on the numerical values of the blank control, the cell adhesion rate can be calculated. The calculation formula is as follows.

[0070]

Number

[0071] As a result, as shown in Figure 8 and as can be seen from the comparison, the recombinant type II humanized collagen of the present invention has better biological adhesion activity than bovine type I collagen (B col I), that is, recombinant type II humanized collagen HC2B-A10 > bovine type I collagen. Unexpectedly, the inventors of the present invention have proved that for HC2B-A10, when the number of repeat sequences is 8, the recombinant type II humanized collagen can have a molecular weight that is easily purified and stable, and can guarantee excellent biological adhesion activity.

[0072] Example 3 Mass spectrometry detection of recombinant type II humanized collagen HC2B-A10 Experimental method

Table 1

[0073] ​The protein sample was reduced with DTT and alkylated with iodoacetamide, and then trypsin was added for overnight enzymatic digestion. The peptide fragments obtained by enzymatic digestion were further desalted with a C18 ZipTip, and then mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) and plated. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometer MALDI-TOF / TOF Ulraflextreme TM , Brucker, Germany was used for analysis (for the technology of peptide mass fingerprinting, refer to Protein J. 2016; 35: 212-7).

[0074] Data search is processed from the MS / MS Ion Search page on the local mascot site. The protein identification results are obtained from the primary mass spectrum of the peptide fragments generated after enzymatic digestion. For the detection parameters, in trypsin enzymatic digestion, two uncleaved sites are set. The alkylation of cysteine is set as a fixed modification. The oxidation of methionine is set as a variable modification. The database for identification is NCBprot.

[0075] Table 1 Molecular weights detected by mass spectrometry of HC2B-A10 and corresponding polypeptides

Table 2

[0076] GTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAKGTPGLPGVKGHRGYPGLDGAK (SEQ ID NO:30) DNA sequence of HC2B-a3 (SEQ ID NO.16): GGTAAACCAGGAGATGATGGAGAAGCAGGAAAACCAGGAAAAGCAGGAGAAAGAGGACCGCCTGGACCGCAAGGAGCACGTGGATTTCCAGGAACCCCGGGACTGCCGGGTGTGAAAGGTCATAGAGGATATCCGGGACTGGATGGAGCAAAAGGAGAAGCAGGGGCACCGGGAGTTAAAGGTGAGAGCGGAAGCCCGGGAGAAAATGGAAGCCCTGGTCCGATGGGTCCGAGAGGGCTGCCGGGTGAAAGAGGGCGTACCGGTCCGGCAGGAGCAGCAGGTGCAAGAGGAAATGATGGACAGCCG, DNA sequence of HC2B-a6 (SEQ ID NO.17): GGAAAACCCGGGAAGGCCGGTGAGCGCGGTCCACCGGGTCCGCAGGGCGCGCGTGGGTTCCCGGGCACCCCGGGTCTGCCGGGTGTTAAAGGTCATCGTGGCAAGCCGGGCAAGGCTGGCGAGCGCGGTCCGCCAGGTCCGCAAGGTGCGAGAGGCTTTCCGGGCACTCCGGGTTTGCCGGGTGTCAAAGGTCACCGTGGTAAACCGGGCAAGGCGGGTGAGCGTGGCCCACCGGGTCCGCAGGGTGCGCGTGGTTTTCCGGGCACGCCGGGTTTGCCGGGCGTTAAAGGCCACCGCGGCAAACCGGGCAAGGCGGGTGAACGTGGCCCACCGGGTCCGCAAGGTGCACGTGGCTTCCCGGGCACCCCGGGTCTGCCTGGCGTGAAAGGTCACCGTGGCAAGCCGGGCAAAGCTGGTGAACGTGGTCCGCCCGGTCCGCAGGGCGCGCGTGGCTTTCCGGGAACCCCGGGCCTGCCGGGCGTTAAGGGTCATCGTGGCAAACCGGGCAAGGCTGGTGAGCGCGGGCCACCGGGTCCTCAAGGTGCCCGTGGCTTCCCGGGCACCCCGGGTCTGCCGGGTGTGAAGGGTCACCGCGGTAAACCGGGCAAGGCAGGCGAACGCGG、 DNA sequence of HC2B-b1 (SEQ ID NO.18): GTGAACCAGGTCGTGAAGGTAGCCCAGGTGCAGATGGACCACCAGGTCGTGATGGTGCAGCAGGAGTGAAAGGAGATCGTGGTGAAACCGGTGCAGTAGGTGCACCTGGTGCGCCAGGTCCGCCAGGTAGCCCTGGTCCAGCCGGTCCTACCGGAAAACAAGGGGATAGAGGAGAAGCAGGAGCACAGGGTCCGATGGGTCCGTCAGGTCCGGCGGGTGCACGTGGTATTCAGGGTCCGCAGGGTCCGCGTGGTGATAAAGGTGAAGCAGGTGAACCGGGGGAAAGAGGATTAAAAGGGCATCGTGGTTTTACGGGTCTGCAGGGTCTGCCTGGTCCGCCTGGTCCGAGCGGTGATCAGGGTGCAAGCGGTCCGGCAGGTCCGAGCGGACCTCGTGGACCTCCGGGTCCTGTGGGTCCTAGTGGTAAGGATGGGGCAAATGGTATTCCTGGTCCTATTGGTCCGCCGGGTCCGCGTGGGAGATCAGGTGAAACCGGACCGGCA、 DNA sequence of HC2B-b3 (SEQ ID NO.19): GGTAGCCCAGGTCCAGCAGGTCCGACAGGTAAACAAGGAGATCGTGGTGAAGCAGGAGCACAAGGACCAATGGGTCCAAGCGGTCCGGCAGGTGCAAGAGGTATTCAAGGGCCGCAAGGGCCACGCGGTGATAAAGGGGAAGCAGGTGAACCAGGTGAGAGAGGGTTAAAAGGACATCGTGGATTTACAGGACTGCAGGGTTTACCAGGTCCGCCGGGACCGAGCGGAGATCAAGGTGCAAGCGGTCCGGCGGGTCCGAGTGGTCCTCGT、 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): GGACCCCAAGGGGCGCGTGGTTTTCCGGGCACCCCGGGCCTGCCGGGCGTGAAGGGTCACCGCGGTTATCCGGGGCTGGATGGCGCGAAGGGTCCGCAAGGTGCTCGCGGTTTCCCGGGAACCCCGGGCCTGCCGGGCGTTAAAGGTCATCGTGGTTATCCGGGTCTGGATGGTGCGAAGGGCCCACAGGGTGCGCGTGGTTTCCCGGGCACCCCTGGGCTCCCGGGTGTGAAAGGTCACAGAGGTTACCCGGGCCTGGATGGTGCCAAAGGTCCGCAAGGTGCGCGTGGTTTTCCGGGCACGCCGGGCTTGCCGGGTGTTAAAGGTCACCGTGGTTATCCGGGTCTGGACGGCGCAAAGGGTCCTCAGGGTGCACGCGGCTTCCCGGGTACGCCGGGCCTTCCGGGCGTGAAGGGCCACCGCGGTTATCCGGGTTTGGACGGTGCCAAGGGTCCGCAGGGTGCGCGTGGATTCCCGGGAACCCCAGGCCTGCCAGGTGTGAAGGGCCACCGTGGCTACCCGGGCTTGGACGGTGCTAAAGGACCGCAGGGCGCACGTGGCTTTCCGGGTACCCCGGGCCTGCCTGGCGTCAAGGGCCATCGTGGTTACCCGGGCCTGGATGGTGCAAAAGGCCCACAGGGTGCCCGTGGTTTCCCGGGTACTCCGGGCCTGCCGGGCGTAAAAGGCCATCGCGGTTACCCGGGTTTGGATGGCGCTAAAGGCCCACAAGGTGCTAGAGGGTTCCCTGGCACCCCGGGTCTGCCGGGCGTTAAAGGCCATCGTGGTTACCCGGGCTTGGACGGCGCGAAGGGTCCGCAAGGCGCGCGTGGTTTTCCGGGTACCCCGGGTCTGCCAGGCGTTAAGGGTCACCGCGGTTACCCGGGTTTAGACGGTGCGAAA 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): GGGGAAAGGGGACTCAAAGGTCACCGCGGTTTCACGGGCCTTCAAGGTCTGCCGGGTCCTCCGGGTCCGAGCGGCGACCAAGGTGCGTCTGGCCCAGCGGGTGAGCGTGGTTTAAAAGGCCACCGCGGTTTCACCGGCCTGCAGGGTTTACCGGGTCCGCCTGGCCCGAGCGGTGATCAAGGTGCAAGCGGCCCGGCAGGCGAACGCGGTCTG、 AAAGGCCATAGAGGTTTTACCGGCCTGCAGGGCTTGCCGGGCCCGCCGGGCCCGAGTGGCGATCAAGGTGCTTCCGGCCCGGCGGGTGAACGTGGCCTGAAGGGCCATCGTGGCTTTACCGGTCTGCAGGGCCTGCCAGGTCCGCCGGGCCCGTCTGGCGACCAGGGTGCGAGCGGTCCAGCCGGTGAGCGCGGCTTGAAGGGCCACCGCGGCTTTACGGGTTTGCAAGGTCTGCCTGGTCCGCCGGGCCCGTCAGGCGATCAAGGGGCGAGCGGCCCGGCGGGTGAACGTGGTCTGAAAGGTCATCGTGGATTCACCGGCCTGCAGGGTCTGCCGGGCCCGC、 CCGGCCCGTCCGGTGACCAGGGTGCTTCCGGTCCGGCTGGTGAGCGCGGTCTTAAGGGACACCGTGGCTTCACCGGTCTGCAGGGCTTGCCGGGTCCTCCGGGGCCGAGCGGCGACCAGGGTGCGTCCGGCCCGGCGGGTGAACGTGGTCTGAAGGGCCACCGTGGTTTTACCGGCTTGCAAGGTCTGCCGGGTCCACCGGGTCCGTCTGGCGATCAGGGAGCCAGCGGTCCGGCAGGCGAGCGTGGTTTGAAGGGTCACCGTGGATTCACCGGCCTGCAGGGGCTGCCGGGCCCGCCGGGTCCGTCGGGCGATCAGGGAGCGAGCGGTCCGGCAGGTGAGCGTGGTCTGAAAGGCCATCGTGGCTTCACTGGTTTGCAAGGCCTGCCTGGCCCACCGGGTCCGAGCGGTGACCAGGGTGCCAGCGGTCCGGCT、 DNA sequence of HC2B-B9 (SEQ ID NO.28): GGAGAAAGGGGGCTTAAGGGCCATCGTGGCTTCACGGGTTTGCAGGGCCTGCCGGGCCCACCGGGTCCGAGCGGCGATCAAGGCGAGCGGGGCCTGAAAGGTCATCGTGGCTTTACCGGTTTGCAGGGCCTGCCGGGCCCACCGGGACCGAGCGGTGATCAGGGGGAGCGTGGTCTGAAAGGTCATCGTGGTTTTACCGGTCTGCAGGGTTTGCCGGGTCCTCCGGGTCCGTCTGGCGACCAAGGTGAGCGTGGTCTGAAAGGTCATCGTGGTTTCACCGGTTTACAAGGCCTGCCGGGCCCACCGGGTCCGAGCGGTGACCAAGGCGAGCGTGGCCTGAAAGGCCACCGCGGTTTCACCGGTCTTCAGGGTCTGCCGGGCCCACCGGGTCCCAGCGGTGACCAGGGTGAACGCGGTCTGAAGGGTCACAGAGGCTTCACCGGTCTCCAAGGCTTACCGGGTCCGCCGGGCCCGAGCGGTGATCAGGGCGAACGTGGTCTGAAGGGCCACCGTGGCTTTACGGGTCTGCAAGGCCTGCCGGGTCCGCCGGGTCCGTCGGGCGACCAGGGTGAGCGCGGTTTGAAGGGCCACCGTGGATTCACCGGCCTGCAGGGCCTGCCGGGACCGCCTGGTCCGTCAGGTGATCAGGGTGAGCGCGGTCTCAAGGGTCATCGTGGTTTTACCGGTTTACAAGGTTTGCCGGGTCCGCCTGGCCCGTCTGGTGATCAAGGCGAACGTGGTCTGAAAGGCCACCGCGGCTTCACTGGTCTGCAGGGCTTGCCGGGCCCACCGGGTCCGTCCGGCGATCAAGGCGAACGTGGTCTGAAGGGTCACCGCGGATTCACGGGCCTGCAGGGCCTGCCGGGTCCGCCAGGTCCGTCCGGCGACCAGGGCGAACGCGGGCTGAAAGGTCACCGTGGCTTTACCGGTTTGCAAGGACTGCCGGGTCCGCCGGGCCCGAGCGGCGACCAG、 DNA sequence of HC2B-B10 (SEQ ID NO.29):

Claims

1. Collagen comprising a plurality of repeating units containing an amino acid sequence shown in SEQ ID NO. 15, 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, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, wherein each repeating unit is directly linked or separated by one or more amino acid residues, preferably the number of repeating units is from 1 to 20, preferably the number of repeating units is from 6 to 10, such as 7, 8 or 9, preferably the collagen comprises an 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 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% or 99% identity thereto, preferably the collagen is recombinant type II humanized collagen.

2. A polynucleotide encoding the collagen according to claim 1, preferably comprising the nucleotide sequence shown in SEQ ID NO.

29.

3. A nucleic acid comprising the polynucleotide according to claim 2, optionally further comprising a nucleotide encoding a purification tag, such as a His tag, GST tag, MBP tag, SUMO tag or NusA tag, and optionally further comprising a nucleotide encoding a leader sequence.

4. A vector comprising the polynucleotide according to claim 2 or the nucleic acid according to claim 3, preferably an expression vector, preferably comprising an expression control element, such as a promoter, terminator and / or enhancer, operably linked to the polynucleotide or nucleic acid.

5. A host cell comprising the polynucleotide according to claim 2, the nucleic acid according to claim 3 or the vector according to claim 4, preferably a bacterium, fungus or animal cell, preferably the bacterium is Escherichia coli, preferably the fungus is yeast, such as Saccharomyces cerevisiae.

6. Step (1) of culturing the host cell according to claim 5 under appropriate culture conditions; Step (2) of harvesting a host cell and / or a culture medium containing collagen; Step (3) of purifying collagen; A method for producing collagen according to claim 1, comprising the above steps.

7. A composition comprising the collagen according to claim 1, preferably a kit, preferably one or more of a biological coating material, a human biomimetic material, a plastic and aesthetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration material, a liver tissue material, a blood vessel repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a pharmaceutical adjuvant material, and a food additive, preferably an injectable composition or an oral composition.

8. The composition according to claim 7, which is a composition for cartilage repair, preferably an injectable composition for cartilage repair.

9. A method for enhancing cell adhesion, comprising the step of contacting cells with the collagen according to claim 1, the polynucleotide according to claim 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5, and / or the composition according to claim 7 or 8.

10. Use in the manufacture of a kit for enhancing cell adhesion or cartilage repair of the collagen according to claim 1, the polynucleotide according to claim 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5, and / or the composition according to claim 7 or 8, or use in one or more of a biological coating material, a human biomimetic material, a plastic and aesthetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration material, a liver tissue material, a blood vessel repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a pharmaceutical adjuvant material, and a food additive.

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