Recombinant humanized XXVII type collagen and application thereof

Abstract

The invention provides recombinant humanized XXVII type collagen and application thereof, and belongs to the technical field of biosynthesis. The recombinant humanized XXVII type collagen has strong cell adhesion and good biocompatibility, can be used for preparing biological dressings, tissue engineering materials, cosmetic fillers, organoid culture scaffolds, cosmetics and the like, and has wide application prospects in the aspects of promoting cell adherence and tissue repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosynthesis, and particularly relates to recombinant humanized collagen XXVII and application. BACKGROUND

[0002] Collagen is a kind of protein widely distributed in human connective tissue, and is also the most abundant protein in human body, which can account for 25% to 35% of the total amount of protein. At present, at least 28 subtypes of collagen have been found in human body, which are located in different tissues and organs.

[0003] COLXXVIIA1 is a member of the collagen fibril gene family and is expressed in cartilage tissue. To begin to understand its role in skeletogenesis, the temporal and spatial distribution of collagen XXVII in developing human skeletal tissue indicates that its gene expression occurs throughout the growth plate and is higher in the resting and proliferative zones than in hypertrophic cartilage. Immunohistochemical analysis shows that collagen XXVII is most obvious in primary ossification centers and hypertrophic cartilage of the growth plate, and accumulates in the pericellular matrix. The synthesis of collagen XXVII overlaps with that of collagen X, which is a marker of chondrocyte hypertrophy, precedes the transformation of cartilage to bone, and is related to cartilage calcification. Extracellular matrix components extracted from mouse meniscus showed by electron microscopy that collagen XXVII is a component of long ribbon-like fibrous structures, filamentous networks, and fine ribbon-like fibrils. Studies have shown that collagen XXVII plays a role in cartilage calcification and the transformation of cartilage to bone.

[0004] However, the current method of producing collagen XXVII is limited, which restricts the research and development of collagen XXVII. Traditional methods such as animal cartilage impurity removal and enzymatic extraction can hardly extract collagen XXVII, which is not conducive to large-scale production. In addition, the immunogenicity of animal-derived collagen cannot be eliminated, which is an important reason for the limitation of collagen in application.

[0005] Therefore, there is an urgent need for a recombinant humanized collagen XXVII that can be produced on a large scale and does not cause an immunogenic reaction, so as to be used as a human structural material for cartilage repair. SUMMARY

[0006] In view of the current needs, the present application uses synthetic biology and structural biology techniques to develop a recombinant humanized collagen XXVII that has good biological activity and can play the function of human collagen.

[0007] The present application provides a recombinant humanized collagen XXVII, which comprises one or more repeating units, wherein the repeating unit comprises:

[0008] (1) The amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or

[0009] (2) An amino acid sequence in which one or more amino acid residues have been mutated in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or

[0010] (3) It has at least 80% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3;

[0011] The number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; each repeating unit is directly connected by peptide bonds or by a linker of one or more amino acids.

[0012] In some specific embodiments of the present invention, mutation is substitution, insertion, deletion or addition.

[0013] In this invention, the recombinant humanized type XXVII collagen comprises:

[0014] (1) The amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; or

[0015] (2) Has at least 80% identical amino acid sequence to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; or

[0016] (3) An amino acid sequence in which one or more amino acid residues have been mutated in the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0017] In some specific embodiments of the present invention, mutation is substitution, insertion, deletion or addition.

[0018] This invention provides a fusion protein comprising the recombinant humanized type XXVII collagen described herein and a purified tag and / or precursor.

[0019] In some specific embodiments of the present invention, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

[0020] This invention provides polynucleotides that encode the collagen or fusion protein described herein.

[0021] In some specific embodiments of the present invention, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 or a degenerate sequence thereof.

[0022] This invention provides a vector comprising the polynucleotides described herein.

[0023] In some specific embodiments of the present invention, the vector comprises a nucleotide encoding a purification tag, a nucleotide encoding a leader, and / or a regulatory element.

[0024] The present invention provides a host cell comprising the polynucleotides described herein, or the vectors described herein.

[0025] In some specific embodiments of the present invention, the host cell is a bacterium, fungus, or animal cell.

[0026] In some specific embodiments of the present invention, the bacteria include Escherichia coli.

[0027] In some specific embodiments of the present invention, the fungus includes yeast.

[0028] This invention provides a method for producing the recombinant humanized type XXVII collagen described herein, comprising the following steps:

[0029] (1) Culture the host cells described in this article;

[0030] (2) Obtaining host cells and / or culture medium containing the recombinant humanized type XXVII collagen; and

[0031] (3) Purify the recombinant humanized type XXVII collagen.

[0032] In some specific embodiments of the present invention, the purification steps include: (1) crude purification of recombinant humanized type XXVII collagen on a Ni affinity chromatography column; (2) enzyme digestion; and / or (3) fine purification of recombinant humanized type XXVII collagen on an ion exchange column.

[0033] This invention provides a trimer collagen comprising the recombinant humanized type XXVII collagen described herein.

[0034] The present invention provides compositions comprising one or more of the following: recombinant humanized type XXVII collagen, fusion protein, polynucleotide, carrier, host cell, and trimer collagen.

[0035] In some specific embodiments of the present invention, the composition is one or more of the following: biological dressings, human biomimetic materials, plastic or cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0036] This invention provides the use of the recombinant humanized type XXVII collagen, fusion protein, polynucleotide, carrier, host cell and / or trimer collagen described herein in the preparation of one or more of the following: bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0037] This invention provides the use of the recombinant humanized type XXVII collagen, fusion protein, trimer collagen and / or compositions described herein in the preparation of medicaments or kits for promoting cell adhesion or cell fixation.

[0038] This invention provides the use of the recombinant humanized type XXVII collagen described herein in the preparation of products for promoting cartilage repair.

[0039] The beneficial effects of this invention are:

[0040] This invention provides a novel core functional region for recombinant humanized type XXVII collagen, and successfully constructs a recombinant humanized type XXVII collagen with good biological activity based on this novel core functional region. The amino acid composition of the collagen protein is 100% identical to the corresponding portion of the amino acid sequence of natural human type XXVII collagen, and its application in humans will not cause immune rejection or allergic reactions. Furthermore, the recombinant humanized type XXVII collagen of this invention exhibits excellent cell adhesion activity, strong biocompatibility, and can be used for tissue repair. Attached Figure Description

[0041] Figure 1 Electrophoresis image of recombinant humanized type XXVII collagen;

[0042] Figure 2 The attached diagram shows 3T3 / NIH cell adhesion.

[0043] Figure 3 The viability of C28 / I2 chondrocytes. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0045] As used in this article, "recombinant humanized collagen" is a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of human collagen.

[0046] In this article, collagen includes both single peptide chains and trimers assembled from them. The trimer specifically refers to a complex composed of three identical single peptide chains, typically forming a triple helix structure.

[0047] As used herein, "fusion protein" refers to a fusion of a protein comprising recombinant humanized type XXVII collagen and another functional portion. In some embodiments, the other functional portion is a protein for promoting collagen secretion, separation, and / or purification. Proteins for promoting collagen secretion, separation, and / or purification are well known to those skilled in the art and include, but are not limited to, restriction enzyme site sequences, signal peptides, and purification tags.

[0048] As used herein, "polynucleotide" refers to a plurality of nucleotides linked by nucleotide bonds (e.g., phosphodiester bonds). The polynucleotides described herein may contain sequences encoding the collagen of this invention. For ease of subsequent processing of the collagen, the polynucleotide may also contain sequences encoding purification tags (e.g., His tags, GST tags, MBP tags, SUMO tags, or NusA tags), and, when necessary, nucleotide sequences encoding leader sequences.

[0049] As used herein, "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0050] As used in this article, a "host cell" is a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques. Host cells can be bacterial, fungal, or animal cells. For example, bacteria can be *Escherichia coli*; fungi include yeasts, such as *Saccharomyces cerevisiae*.

[0051] This invention provides recombinant humanized type XXVII collagen, comprising one or more repeating units, wherein the repeating unit comprises:

[0052] (1) The amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or

[0053] (2) An amino acid sequence in which one or more amino acid residues have been mutated in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or

[0054] (3) It has at least 80% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3;

[0055] The number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; each repeating unit is directly connected by peptide bonds or by a linker of one or more amino acids.

[0056] In some implementations, mutations are substitutions, insertions, deletions, or additions.

[0057] In some implementations, the mutation can be a substitution, such as a conserved amino acid substitution.

[0058] In some embodiments, the repeating unit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or any percentage identity between 80% and 99% with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0059] In some implementations, the number of repeating units is 9.

[0060] In some implementations, the repeating units are directly connected.

[0061] In some embodiments, each repeating unit is spaced apart by one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid residues.

[0062] In some embodiments, the recombinant humanized type XXVII collagen of the present invention is synthetic or recombinantly expressed.

[0063] In this invention, the recombinant humanized type XXVII collagen can be prepared into a composition or a kit.

[0064] In some embodiments, the composition or kit may be a composition or kit for promoting cell adhesion or cell fixation.

[0065] In some embodiments, the composition or kit may be a composition or kit for tissue filling and / or volume enhancement.

[0066] In some embodiments, the composition or kit may also contain auxiliary substances, such as buffers.

[0067] In some embodiments, the compositions of the present invention may be cartilage repair agents comprising the recombinant humanized type XXVII collagen described herein.

[0068] The compositions of the present invention may be injectable. The compositions of the present invention may be human structural materials, for example, used for cartilage repair, and do not elicit an immune response in the human body.

[0069] The present invention also provides a method for constructing recombinant humanized type XXVII collagen, including functional region screening and strain construction, large-scale bio-fermentation, protein induction expression, purification and optional enzymatic digestion steps.

[0070] In some embodiments, the functional region screening and strain construction steps include: (1) large-scale functional region screening to obtain the target gene functional region; (2) inserting the obtained target gene functional region into an expression vector (e.g., PET-28a-Trx-His) to obtain a recombinant expression plasmid; (3) transforming the recombinant expression plasmid into competent Escherichia coli cells (e.g., BL21(DE3)) and screening to obtain positive Escherichia coli genetically engineered bacteria.

[0071] In some embodiments, the large-scale bio-fermentation step includes: adding the screened positive Escherichia coli genetically engineered bacteria to a shake flask containing antibiotic stock solution and culturing it in a constant temperature shaker at 220 rpm and 37°C.

[0072] In some embodiments, the steps of inducing protein expression include: (1) cooling the cultured shake flask to 16-30°C; (2) adding IPTG stock solution to induce expression; and (3) collecting the bacterial cells after inducing expression by placing the bacterial culture in a centrifuge bottle and centrifuging at 6000 rpm and 4°C for 12 min.

[0073] In some embodiments, the purification and optional enzymatic digestion steps of recombinant humanized type XXVII collagen include: (1) crude purification of recombinant humanized type XXVII collagen on a Ni affinity chromatography column; (2) enzymatic digestion with TEV enzyme added in a certain proportion; and (3) purification of recombinant humanized type XXVII collagen on an ion exchange column.

[0074] The following embodiments are provided to illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.

[0075] Example 1: Screening of functional regions of type XXVII collagen

[0076] Large-scale functional region screening of natural human type XXVII collagen yielded the following different protein functional regions.

[0077] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP (SEQ ID NO:1)

[0078] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP (SEQ ID NO:2)

[0079] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP (SEQ ID NO:3)

[0080] The amino acid fragments in these regions were optimized by repeating them n times and directly linking them to obtain recombinant collagen rhColXXVII-1, rhColXXVII-2 and rhColXXVII-3, and their corresponding amino acid sequences are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0081] rhColXXVII-1:

[0082] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0083] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0084] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0085] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0086] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0087] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0088] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0089] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP

[0090] GLPGLFGLPGSDGERGLPGVPGKRGKMGMP(SEQ ID NO:4)

[0091] rhColXXVII-2:

[0092] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0093] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0094] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0095] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0096] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0097] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0098] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0099] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP

[0100] GPPGPQGRPGRPGQQGVAGERGHLGSRGFP(SEQ ID NO:5)

[0101] rhColXXVII-3:

[0102] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0103] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0104] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0105] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0106] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0107] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0108] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0109] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP

[0110] GKQGEKGRTGAKGAKGYQGQLGEMGVPGDP (SEQ ID NO:6)

[0111] The nucleotide sequences corresponding to the above amino acid sequences are as follows:

[0112] The nucleotide sequence corresponding to rhColXXVII-1 is as follows:

[0113] GGACTACCCGGGCTCTTCGGCCTGCCGGGCTCCGACGGCGAGCGTGGCCTGCCTGGTGTTCCGGGCAAGCGCGGTAAGATGGGTATGCCGGGGCTGCCGGGCTTGTTTGGTCTGCCGGGTAGCGATGGTGAACGTGGTCTGCCGGGTGTTCCGGGAAAGCGCGGTAAAATGGGTATGCCGGGTCTGCCGGGCTTGTTTGGCCTTCCGGGTTCCGACGGCGAACGTGGTTTGCCAGGCGTTCCGGGTAAACGTGGTAAAATGGGTATGCCGGGCCTGCCGGGTTTGTTCGGCTTACCGGGCAGCGACGGCGAGCGCGGTCTACCGGGCGTGCCGGGCAAGCGTGGTAAAATGGGTATGCCAGGCCTGCCGGGTCTGTTCGGTCTGCCGGGTAGCGATGGTGAGCGTGGCCTGCCGGGGGTGCCTGGCAAACGTGGTAAGATGGGCATGCCGGGTCTCCCGGGCTTGTTTGGTTTGCCAGGCTCGGATGGCGAGCGCGGTCTGCCGGGCGTTCCGGGTAAACGTGGTAAAATGGGTATGCCGGGCCTGCCGGGTCTGTTCGGCCTGCCGGGTAGCGATGGTGAGAGAGGCCTGCCGGGCGTGCCGGGTAAGCGCGGTAAAATGGGCATGCCGGGTCTGCCAGGTTTGTTTGGCCTGCCGGGCTCTGACGGCGAACGTGGTCTGCCGGGTGTCCCAGGCAAGCGCGGGAAGATGGGTATGCCTGGCTTGCCGGGCCTTTTCGGCCTGCCGGGTAGCGACGGCGAACGTGGTCTGCCGGGTGTGCCGGGTAAACGTGGTAAGATGGGCATGCCC (SEQ ID NO:7)

[0114] The nucleotide sequence corresponding to rhColXXVII-2 is as follows:

[0115] GGTCCACCCGGACCGCAGGGTCGTCCGGGTCGTCCGGGACAACAGGGTGTCGCAGGTGAACGCGGTCATCTGGGCTCCCGTGGTTTCCCGGGACCGCCGGGCCCGCAGGGCCGTCCGGGTCGCCCGGGCCAACAAGGCGTTGCGGGTGAGCGTGGTCATCTGGGCTCTCGCGGTTTTCCGGGCCCACCGGGCCCTCAAGGTCGTCCGGGTCGTCCGGGACAGCAGGGCGTGGCGGGTGAACGTGGTCACTTAGGCAGCCGTGGATTCCCGGGCCCACCGGGTCCACAAGGCCGTCCGGGGCGTCCGGGTCAACAAGGTGTTGCCGGTGAACGTGGCCACCTGGGTAGCAGAGGCTTCCCGGGCCCACCGGGCCCGCAAGGTCGCCCGGGCCGCCCGGGCCAGCAGGGTGTTGCGGGTGAGCGCGGCCATCTGGGTTCGCGAGGTTTTCCGGGCCCGCCGGGCCCGCAGGGCCGTCCGGGTAGACCTGGCCAGCAAGGGGTGGCTGGCGAGCGCGGTCACTTGGGTTCCCGTGGCTTCCCGGGCCCTCCGGGTCCGCAGGGTCGCCCAGGCCGCCCTGGTCAGCAGGGCGTGGCTGGCGAGCGTGGTCACCTGGGGAGCCGTGGTTTTCCGGGTCCTCCGGGTCCGCAGGGTCGTCCGGGCCGTCCGGGCCAACAAGGCGTGGCAGGTGAACGCGGTCATCTGGGTAGCCGTGGTTTTCCGGGTCCGCCTGGTCCGCAGGGCCGTCCGGGCCGTCCGGGTCAGCAAGGTGTTGCGGGTGAGCGCGGTCACTTGGGTAGCCGTGGCTTCCCG (SEQ ID NO:8)

[0116] The nucleotide sequence corresponding to rhColXXVII-3 is as follows:

[0117] GGAAAACAAGGGGAGAAGGGTCGTACGGGTGCCAAGGGTGCAAAGGGCTACCAGGGTCAACTGGGTGAGATGGGTGTGCCGGGCGATCCGGGTAAACAAGGCGAAAAAGGACGCACTGGTGCGAAAGGCGCGAAGGGCTACCAGGGCCAGTTGGGTGAAATGGGCGTGCCGGGTGATCCGGGTAAACAGGGTGAGAAGGGCAGAACGGGCGCTAAGGGGGCTAAGGGCTATCAGGGTCAGTTAGGCGAGATGGGCGTTCCGGGCGACCCGGGCAAACAGGGCGAGAAAGGCCGCACCGGTGCAAAGGGCGCGAAAGGCTACCAAGGTCAACTGGGCGAAATGGGCGTGCCTGGCGACCCGGGTAAACAGGGCGAGAAAGGTCGTACCGGCGCTAAGGGTGCGAAGGGTTATCAGGGCCAGCTGGGTGAAATGGGTGTTCCGGGTGATCCGGGTAAACAGGGTGAAAAAGGCCGTACCGGTGCAAAGGGCGCGAAAGGCTACCAGGGCCAACTGGGTGAGATGGGTGTCCCGGGTGACCCGGGCAAGCAAGGCGAAAAAGGCCGCACCGGTGCCAAGGGTGCGAAGGGCTATCAAGGTCAATTGGGTGAGATGGGTGTGCCGGGTGATCCGGGCAAACAGGGTGAGAAAGGTCGTACCGGTGCGAAAGGCGCGAAGGGCTACCAAGGTCAGCTCGGTGAAATGGGCGTTCCGGGTGACCCAGGCAAACAAGGGGAAAAGGGTCGTACCGGCGCGAAGGGTGCCAAGGGTTATCAGGGTCAACTGGGTGAAATGGGTGTTCCAGGTGACCCG (SEQ ID NO:9)

[0118] Example 2: Preparation and Expression of Recombinant Humanized Collagen Type XXVII

[0119] Insert the gene functional region synthesized in Example 1 into the pET-28a-Trx-His expression vector to obtain the corresponding recombinant expression plasmid.

[0120] The successfully constructed expression plasmid was transformed into Escherichia coli competent cells BL21(DE3). The specific process was as follows: (1) The Escherichia coli competent cells BL21(DE3) were taken out of the ultra-low temperature freezer and placed on ice. When they were half-thawed, 2 μl of the plasmid to be transformed was added to the Escherichia coli competent cells BL21(DE3) and mixed slightly 2-3 times. (2) The mixture was placed on ice for 30 min, and then heat-shocked in a water bath at 42℃ for 45-90 s. After taking it out, it was placed on ice for 2 min. (3) It was transferred to a biosafety cabinet and 700 μl of liquid LB medium was added. Then it was cultured at 37℃ and 220 rpm for 60 min. (4) 200 μl of bacterial solution was evenly spread on LB plates containing kanamycin sulfate. (5) The plates were cultured in an incubator at 37℃ for 15-17 h until uniform colonies grew.

[0121] Pick 5-6 single colonies from the transformed LB agar plates and place them in a shake flask containing antibiotic stock solution (kanamycin 100 mg / L). Incubate at 220 rpm and 37°C in a constant temperature shaker for a certain period of time until the colonies appear as a mist. Then, cool the shake flasks to 16-30°C, add IPTG (0.5 mM) to induce expression for a period of time, aliquot the bacterial culture into centrifuge bottles, centrifuge at 6000 rpm and 4°C for 12 min, collect the bacterial cells, record the cell weight, and perform electrophoresis analysis.

[0122] The collected bacterial cells were resuspended in a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). The bacterial suspension was cooled to ≤15℃ and homogenized twice or sonicated to disrupt the cells. After the cell disruption was completed, the bacterial suspension was collected. The disrupted bacterial suspension was aliquoted into centrifuge bottles and centrifuged at 17000 rpm and 4℃ for 30 min. The supernatant was collected.

[0123] The recombinant humanized type XXVII collagen was purified and enzymatically digested. The specific process was as follows:

[0124] (1) Crude Purification: a. Equilibrate the column: Equilibrate the column with equilibration buffer (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) at a flow rate of 10 mL / min. b. Load the sample: Add the supernatant after centrifugation to the column until the liquid has completely flowed out, at a flow rate of 5 mL / min. c. Wash away contaminating proteins: Add 100 mL of washing buffer (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid has completely flowed out, at a flow rate of 10 mL / min. d. Collect the target protein: Add 20 mL of elution buffer (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) at a flow rate of 10 mL / min, collect the flow-through, and perform electrophoresis. e. Wash the column with 1 M imidazole working solution at a flow rate of 10 mL / min.

[0125] (2) Enzyme digestion: Add TEV enzyme at a ratio of total protein to total TEV enzyme of 20:1 and digest at 16℃ for 2 h. Place the digested protein solution into a dialysis bag and dialyze at 4℃ for 2 h, then transfer it to a new dialysis solution (20 mM sodium chloride, 20 mM Tris) and dialyze overnight at 4℃.

[0126] (3) Purification: a. Column equilibration: Equilibrate the column with solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 mL / min. b. Sample loading: Load the sample at a flow rate of 5 mL / min and collect the flow-through sample. Perform electrophoresis and store the protein at 4°C. c. Elution: Wash the column with solution B (1 M sodium chloride, 20 mM Tris) for 5 CVs. d. Wash the column. SDS-PAGE analysis of purified recombinant humanized type XXVII collagen.

[0127] In the electrophoretic analysis of recombinant humanized type XXVII collagen rhColXXVII (1-3), the molecular weight of the target protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The theoretical molecular weights calculated based on the amino acid sequence were 26274.98, 26534.21, and 26489.46 Da, respectively. The actual electrophoretic detection results showed (e.g.) Figure 1 As shown in the figure, the molecular weight corresponding to the migration position of the target band deviates from the theoretical value. This phenomenon is due to the positive / negative charge of collagen itself, which leads to insufficient SDS binding, ultimately manifesting as a deviation in apparent molecular weight, and is a normal phenomenon.

[0128] The purification process employed a two-step chromatographic strategy: first, the histidine-tagged fusion protein was captured by nickel column affinity chromatography; then, the tag sequence was removed by enzymatic digestion; and finally, fine purification was performed using ion chromatography. Electrophoresis patterns showed that the final product, stained with Coomassie Brilliant Blue, exhibited minimal background contaminating protein bands.

[0129] The above results confirm that: 1) the recombinant expression system successfully produced recombinant humanized type XXVII collagen; 2) the chromatographic purification protocol effectively separated the target protein from host cell impurities; and 3) the molecular weight was consistent with the theoretical molecular weight, with the deviation within an acceptable range. The biological activity of the expressed and purified collagen will be verified below.

[0130] Example 3: Cell adhesion experiment of recombinant humanized type XXVII collagen

[0131] The specific experimental method is as follows:

[0132] (1) The concentration of the protein samples to be tested was detected by ultraviolet absorption method, including bovine type I collagen (China National Institutes for Food and Drug Control, No.: 380002) and the collagen rhCol-XXVII provided in this invention. The ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated using the empirical formula C(μg / mL) = 144 × (A215 - A225), where C represents the protein concentration in the sample to be tested, A215 represents the absorbance of the sample to be tested at a wavelength of 215 nm, and A225 represents the absorbance of the sample to be tested at a wavelength of 225 nm. Note that the detection should be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues that are not colorimetric by Coomassie Brilliant Blue. After the protein concentration was measured, the concentration of all the proteins to be tested was adjusted to 0.5 mg / mL with PBS.

[0133] (2) Add 100 μL of various protein solutions and blank PBS solution control (NC) to a 96-well plate and let it stand at room temperature for 60 min.

[0134] (3) Add 10 to each hole 5 A well-cultured 3T3 / NIH cell was incubated at 37°C for 60 minutes.

[0135] (4) Wash each well with PBS 4 times.

[0136] (5) The absorbance at OD450nm was measured using the CCK8 assay kit (Beyotime, product catalog number C0038). The cell adhesion rate could be calculated based on the values ​​of the blank control. The calculation formula is as follows: Cell adhesion rate = {(test wells - blank wells) / (positive wells - blank wells)} × 100%. The cell adhesion rate reflects the activity of collagen. The higher the protein activity, the better it can provide a high-quality external environment for cells in a short time, helping cells adhere.

[0137] The results are as follows Figure 2 As shown in the comparison, compared with bovine type I collagen (PC group, 0.5 mg / mL), the humanized collagen rhCol-XXVII of the present invention has better bioadhesion activity on 3T3 / NIH cells, and rhColXXVII-1 and rhColXXVII-2 have higher fibroblast adhesion activity than rhColXXVII-3.

[0138] Example 4: Cell activity experiment of recombinant humanized type XXVII collagen on chondrocyte C28 / I2 cells.

[0139] Type XXVII collagen is expressed in cartilage tissue. Its gene expression occurs throughout the growth plate, and is higher in resting and proliferating zones than in hypertrophic cartilage. Type XXVII collagen is most prominent in ossification centers and hypertrophic cartilage of the growth plate, and accumulates in the pericellular matrix. Its synthesis precedes the conversion of cartilage to bone and is associated with cartilage calcification. To clarify the activity of the purified rhColXXVII in chondrocytes, this invention demonstrates its pro-activation effect on C28 / I2 chondrocytes through this embodiment.

[0140] The specific experimental steps are as follows:

[0141] After resuscitation and passage to adjust cell condition, C28 / I2 cells were seeded into 96-well plates at a density of 100 μL per well (10,000 cells / well). Cells were cultured at 37°C with 5% CO2 for 24 hours. After 24 hours, pre-diluted test samples were added and cultured again at 37°C with 5% CO2 for 24 hours.

[0142] Discard the supernatant and add CCK8 assay reagent (Beyotime, product catalog number C0038) to each well. Read the absorbance at OD450nm using a microplate reader. Cell viability can be calculated based on the values ​​of the blank control. The calculation formula is as follows: Cell viability = {(test wells - blank wells) / (positive wells - blank wells)} × 100%.

[0143] according to Figure 3 The results showed that the three collagen proteins, rhCol-XXVII, exhibited certain biocompatibility with chondrocyte C28 / I2 at concentrations below 100 μg / mL. Compared to the other two, rhColXXVII-3 showed relatively poor biocompatibility with a CC50 value of 76.10 μg / mL. rhColXXVII-1 and rhColXXVII-2 showed no significant cytotoxicity, and both significantly improved chondrocyte viability and promoted chondrocyte growth at their highest concentrations of 250 μg / mL and 400 μg / mL, indicating their potential applications in cartilage defect repair.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Recombinant humanized type XXVII collagen, comprising one or more repeating units, wherein the repeating unit comprises: (1) The amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or (2) An amino acid sequence in which one or more amino acid residues have been mutated in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or (3) It has at least 80% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; The number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; each repeating unit is directly connected by peptide bonds or by a linker of one or more amino acids. Preferably, the mutation is a substitution, insertion, deletion, or addition.

2. The recombinant humanized type XXVII collagen according to claim 1, characterized in that, The recombinant humanized type XXVII collagen comprises: (1) The amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; or (2) Has at least 80% identical amino acid sequence to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; or (3) An amino acid sequence in which one or more amino acid residues have been mutated in the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; preferably, the mutation is substitution, insertion, deletion or addition.

3. A fusion protein comprising the recombinant humanized type XXVII collagen as described in claim 1 or 2 and a purified tag and / or precursor; preferably, the purified tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

4. A polynucleotide encoding the recombinant humanized type XXVII collagen of claim 1 or 2 or the fusion protein of claim 3; preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 or a degenerate sequence thereof.

5. A vector comprising the polynucleotide of claim 4; preferably, the vector comprises a nucleotide encoding a purification tag, a nucleotide encoding a leader, and / or a regulatory element.

6. A host cell comprising the polynucleotide of claim 4 or the vector of claim 5; preferably, the host cell is a bacterium, fungus or animal cell; preferably, the bacterium comprises Escherichia coli; preferably, the fungus comprises yeast.

7. A method for producing the recombinant humanized type XXVII collagen as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Culturing the host cells as described in claim 6; (2) Obtain host cells and / or culture medium containing the recombinant humanized type XXVII collagen; and (3) Purifying the recombinant humanized type XXVII collagen; preferably, comprising: (1) crudely purifying the recombinant humanized type XXVII collagen on a Ni affinity chromatography column; (2) enzyme digestion; And / or (3) Ion exchange column purification of recombinant humanized type XXVII collagen.

8. Trimeric collagen, comprising the recombinant humanized type XXVII collagen of claim 1 or 2.

9. A composition, characterized in that, It comprises one or more of the following: the recombinant humanized type XXVII collagen of claim 1 or 2, the fusion protein of claim 3, the polynucleotide of claim 4, the vector of claim 5, the host cell of claim 6, and the trimer collagen of claim 8.

10. The composition according to claim 9, characterized in that, The composition is one or more of the following: biological dressings, human biomimetic materials, plastic or cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

11. The use of the recombinant humanized type XXVII collagen of claim 1 or 2, the fusion protein of claim 3, the polynucleotide of claim 4, the carrier of claim 5, the host cell of claim 6, and / or the trimer collagen of claim 8 in the preparation of one or more of the following: bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives.

12. Use of the recombinant humanized type XXVII collagen of claim 1 or 2, the fusion protein of claim 3, the trimer collagen of claim 8, and / or the composition of claim 9 in the preparation of a medicament or kit for promoting cell adhesion or cell fixation.

13. Use of the recombinant humanized type XXVII collagen of claim 1 or 2 in the preparation of products for promoting cartilage repair.

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