Recombinant VIII type humanized collagen and application thereof

Abstract

The invention provides recombinant VIII type humanized collagen and application thereof, and the recombinant VIII type humanized collagen is of a triple helix structure, has good cell adhesion activity, does not generate immunological rejection and anaphylactic reaction when being applied to a human body, is a brand new human body synthetic biological material, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and particularly relates to human body structural materials and their biosynthetic preparation methods. Background Art

[0002] Collagen is a type of protein widely distributed in the human body's connective tissue. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. It has been discovered that the human body has at least 28 collagen subtypes, which are located in different tissues and organs.

[0003] Type VIII collagen is a member of the collagen family, primarily secreted by endothelial cells, epithelial cells, and Schwann cells, and distributed throughout the ocular and vascular tissues. Type VIII collagen plays a key role in maintaining corneal transparency and normal structure. It interacts with other types of collagen and other components of the cornea to form the cornea's orderly fibrous network, maintaining a smooth and uniform corneal appearance. This allows light to pass smoothly through the cornea and into the eye, providing the basis for clear vision. Type VIII collagen is also distributed within the lens of the eye, playing a crucial role in maintaining its normal structure and transparency, helping to ensure proper refraction and conduction of light within the lens and maintaining good vision. In vascular tissue, it helps maintain the integrity and stability of the blood vessel wall. It is distributed around endothelial cells and, along with other extracellular matrix components, provides physical support for these cells, enabling them to withstand the pressure of blood flow, preventing rupture or leakage, and ensuring the normal function of the vascular system. Type VIII collagen can also promote the migration and proliferation of endothelial cells, thereby inducing the formation of new blood vessels, bringing oxygen and nutrients to the wound site, transporting metabolic waste, providing the necessary material basis and physiological environment for wound healing, and accelerating the wound repair process.

[0004] Type VIII collagen binds to specific receptors on the cell surface, activating intracellular signaling pathways and thus influencing cell proliferation. During tissue development and repair, it controls the rate and number of cell divisions, ensuring normal tissue growth and effective repair after injury. The interaction between type VIII collagen and cell surface receptors transmits specific signals, guiding cell differentiation in specific directions. For example, during embryonic development, it influences the differentiation of endothelial cells into different types of vascular cells, participating in the formation and development of the vascular system. Type VIII collagen acts as a temporary extracellular matrix, providing a scaffold for cells migrating to the wound site to attach and migrate. This attracts a variety of cells, including fibroblasts and endothelial cells, to the wound site. These cells migrate and proliferate on the scaffold, beginning to synthesize and secrete new extracellular matrix components, laying the foundation for wound healing.

[0005] Until now, most collagen used in various studies has been derived from animal tissues and skin extracts. However, the poor water solubility and processability of animal-derived collagen have directly limited the development of many potential applications. Genetic engineering to produce full-length type VIII collagen also presents various challenges. For example, codon usage differs between prokaryotic and eukaryotic cells. The full-length type VIII collagen gene originates from a eukaryotic organism, and its codon usage may differ from the prokaryotic host bacteria. This can cause ribosomes to pause at rare codons during translation in prokaryotes, reducing protein synthesis efficiency and even impairing the proper folding and elongation of nascent peptide chains. When produced in eukaryotic systems, excessive intracellular accumulation of the recombinant protein can negatively impact the cells, potentially resulting in lower-than-expected expression levels of full-length type VIII collagen. Furthermore, yeast cells may have limited secretion capacity for some large molecular weight proteins, resulting in inefficient extracellular secretion of some proteins. Summary of the Invention

[0006] The present invention utilizes synthetic biology and structural biology techniques to develop a recombinant collagen having a recombinant humanized collagen VIII triple helix structure and capable of performing the functions of human collagen.

[0007] In a first aspect, the present invention provides a recombinant humanized type VIII collagen comprising n repeating units, wherein the repeating units are connected directly or via a linker, and the repeating units comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8 or a variant thereof.

[0008] In some embodiments, the linker comprises one amino acid or multiple amino acid residues; preferably, the linker consists of 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0009] In some embodiments, the variant is (1) an amino acid sequence comprising one or more amino acid substitutions, deletions, or insertions of SEQ ID NO: 2 or SEQ ID NO: 8; or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or SEQ ID NO: 8. In some embodiments, n is an integer from 1 to 20. In some embodiments, n is 4, 8, or 12.

[0010] In some embodiments, the collagen comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.7.

[0011] In some embodiments, the collagen comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 7.

[0012] In some embodiments, the collagen comprises an amino acid sequence obtained by substituting, deleting or inserting one or more amino acids based on SEQ ID NO: 1 or SEQ ID NO: 7.

[0013] The second aspect of the present invention provides a fusion protein comprising the recombinant humanized type VIII collagen according to the first aspect of the present invention and a purification tag and / or a leader.

[0014] In some embodiments, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag.

[0015] The third aspect of the present invention provides a polynucleotide encoding the collagen described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0016] In some embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9 or a degenerate sequence thereof.

[0017] The fourth aspect of the present invention provides a vector comprising the polynucleotide according to the third aspect of the present invention.

[0018] In some embodiments, the vector is an expression vector; in some embodiments, the vector comprises a control element operably linked to the polynucleotide; in some embodiments, the control element is selected from a promoter, a terminator and / or an enhancer.

[0019] The fifth aspect of the present invention provides a host cell comprising the polynucleotide according to the third aspect of the present invention, or the vector according to the fourth aspect of the present invention.

[0020] In some embodiments, the host cell is a bacterial, fungal or animal cell; preferably, the bacteria include Escherichia coli; preferably, the fungus includes yeast, such as Saccharomyces cerevisiae.

[0021] The sixth aspect of the present invention provides a collagen trimer comprising the recombinant humanized type VIII collagen described in the first aspect of the present invention.

[0022] The seventh aspect of the present invention provides a method for producing the recombinant humanized type VIII collagen described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention, the method comprising the following steps:

[0023] (1) culturing the host cell according to the fifth aspect of the present invention under suitable culture conditions;

[0024] (2) harvesting host cells and / or culture medium containing recombinant humanized type VIII collagen or fusion protein; and optionally

[0025] (3) Purifying the recombinant humanized type VIII collagen or fusion protein.

[0026] The eighth aspect of the present invention provides a composition comprising one or more of the recombinant humanized type VIII collagen described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the host cell described in the fifth aspect of the present invention, and the collagen trimer described in the sixth aspect of the present invention.

[0027] In some embodiments, the composition is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives; preferably, the composition is a topical composition, an injectable composition or an oral composition; preferably, the composition is a composition in the form of a solution, a lyophilized powder, a gel, a sponge or a fiber.

[0028] The ninth aspect of the present invention provides the use of the recombinant humanized type VIII collagen described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the host cell described in the fifth aspect of the present invention, or the collagen trimer described in the sixth aspect of the present invention in the preparation of one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone / cartilage regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0029] The tenth aspect of the present invention provides a method for promoting cell adhesion, which includes the step of contacting the recombinant humanized type VIII collagen described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the host cell described in the fifth aspect of the present invention, the collagen trimer described in the sixth aspect of the present invention and / or the composition described in the eighth aspect of the present invention with cells.

[0030] In some embodiments, the cell is an animal cell; in some embodiments, the animal cell is a mammalian cell; in some embodiments, the mammalian cell is a human cell; in some embodiments, the promoting cell adhesion is performed in vitro.

[0031] The eleventh aspect of the present invention provides a method for performing plastic surgery, tissue injection filling, ophthalmic treatment, nerve repair or vascular repair on a subject in need thereof, which comprises administering the recombinant humanized type VIII collagen described in the first aspect of the present invention to the subject, preferably orally or by injection; preferably, the subject is human.

[0032] The twelfth aspect of the present invention provides the use of the recombinant humanized type VIII collagen described in the first aspect of the present invention and / or the collagen trimer described in the sixth aspect of the present invention in the preparation of a medicament for a disease or condition associated with type VIII collagen deficiency, preferably, the disease or condition is anterior segment dysplasia.

[0033] Advantages of the present invention include:

[0034] 1. The recombinant type VIII collagen of the present invention is derived from natural human type VIII collagen and will not cause immune rejection or allergic reactions when applied to the human body.

[0035] 2. The recombinant type VIII collagen of the present invention is suitable for preparation by biosynthesis methods, has a large expression amount and is suitable for subsequent purification, has high production efficiency and is pollution-free, and has good prospects for industrial production.

[0036] 3. The recombinant type VIII collagen of the present invention has higher cell adhesion activity than the control group. Furthermore, as verified by circular dichroism, it also has a stable triple-helical structure and good biological activity. It can be widely used as a human biomaterial, for example, in the fields of chronic wound healing disorders, bone and cartilage damage repair, cardiovascular disease, ophthalmology, dermatology and cosmetic medicine, nervous system repair, degenerative diseases, and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the electrophoresis result of collagen VIII-1.

[0038] Figure 2 This is the electrophoresis result of collagen VIII-2.

[0039] Figure 3 This is the electrophoresis result of collagen VIII-3.

[0040] Figure 4 This is the CD map of collagen VIII-1.

[0041] Figure 5 This is the CD map of collagen VIII-2.

[0042] Figure 6 This is the CD map of collagen VIII-3.

[0043] Figure 7 Figure 2 shows cell adhesion diagrams of three recombinant humanized type VIII collagens. DETAILED DESCRIPTION

[0044] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0045] Definition of terms

[0046] As used in this article, "recombinant collagen" is a new type of biomaterial with the same or similar amino acid sequence as human collagen, which is screened and prepared using cutting-edge structural biology, genetic engineering and other technologies, with the gene encoding the functional region of a specific type of human collagen as a template.

[0047] As used herein, "recombinant humanized type VIII collagen" refers to a recombinant protein consisting of or substantially consisting of a sequence derived from human type VIII collagen. In this context, recombinant humanized type VIII collagen may consist of or substantially consist of a fragment or multiple repeats of a fragment derived from human type VIII collagen.

[0048] As used herein, "one or more" can be any suitable integer. In the case of collagen mutations (e.g., substitutions, deletions, insertions, or additions), "one or more" is a number readily determined by those skilled in the art, such as 1-90 and any integers and ranges therebetween, such as 1, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, etc.

[0049] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0050] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well 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); bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. A vector can contain a nucleic acid of the present invention to facilitate introduction into cells for expression. A vector can contain expression control elements, such as a promoter, terminator, and / or enhancer, operably linked to the nucleic acid.

[0051] As used herein, the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to a control sequence providing for its expression. In this article, the expression vector is an E. coli expression vector.

[0052] As used herein, the term "recombinant expression vector" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not originally found in nature to contain a nucleic acid segment, or is synthetic, and that contains one or more control sequences.

[0053] As used herein, the term "control sequence" means the nucleic acid sequence necessary for expressing the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence can be natural (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or be natural or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the control sequence includes a promoter, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that are conducive to connecting the control sequence to the coding region of the polynucleotide encoding the polypeptide, these control sequences can be provided with a plurality of linkers.

[0054] 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 of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell can be a eukaryotic cell or a prokaryotic cell. For example, a eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell can be an Escherichia coli cell.

[0055] Recombinant humanized type VIII collagen

[0056] The present invention provides a recombinant humanized type VIII collagen protein. The recombinant humanized type VIII collagen protein of the present invention may comprise one or more repeating units, wherein the repeating units comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8 or a variant thereof.

[0057] In some embodiments, the variant comprises an amino acid sequence obtained by mutation (substitution, addition, or deletion) of one or more amino acid residues based on SEQ ID NO: 2 or SEQ ID NO: 8.

[0058] In some embodiments, the variant comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 or SEQ ID NO:8.

[0059] In some embodiments, the number of repeating units can be 1-20.

[0060] In some embodiments, 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.

[0061] In some embodiments, the number of repeating units is 4, 8, or 12.

[0062] In some embodiments, the mutation can be a substitution, such as a conservative amino acid substitution.

[0063] In some embodiments, the repeat units are directly linked.

[0064] In some embodiments, each repeat unit is separated by one or more amino acid residues.

[0065] In some embodiments, when a repeating unit is mutated, the recombinant collagen obtained retains comparable or superior cell adhesion properties compared to collagen obtained from the unmutated repeating unit.

[0066] In some embodiments, the recombinant humanized type VIII collagen of the present invention is synthesized or recombinantly expressed.

[0067] In some embodiments, the collagen comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.7, or an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:7, or an amino acid sequence obtained by substitution, deletion, or insertion of one or more amino acids on the basis of SEQ ID NO:1 or SEQ ID NO:7.

[0068] Recombinant humanized collagen type VIII encoding nucleic acid

[0069] The present invention provides a polynucleotide encoding recombinant type VIII humanized collagen for expression.

[0070] In some embodiments, the polynucleotide is codon-optimized for the host cell in which it is to be expressed.

[0071] In some embodiments, the polynucleotide encoding the recombinant humanized type VIII collagen 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.

[0072] In some embodiments, the polynucleotide encoding recombinant humanized type VIII collagen may further comprise nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag, or nucleotides encoding a leader sequence to facilitate purification or secretion of the polypeptide.

[0073] Composition and application thereof

[0074] The present invention also provides a composition comprising recombinant humanized type VIII collagen. The composition includes, but is not limited to, biological dressings, human biomimetic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filler materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone / cartilage regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives.

[0075] The administration methods of the composition include, but are not limited to, injection, application or oral administration.

[0076] Production of recombinant humanized type VIII collagen

[0077] The present invention also provides the production of recombinant humanized type VIII collagen, including functional region screening and strain construction, large-scale biological fermentation, protein induced expression, purification and optional enzymatic cleavage steps.

[0078] In some embodiments, the functional region screening and strain construction steps include: (1) large-scale functional region screening to obtain the functional region of the target gene; (2) inserting the obtained functional region of the target gene into an expression vector (such as PET-28a-Trx-His) to obtain a recombinant expression plasmid; (3) transferring the recombinant expression plasmid into Escherichia coli competent cells (such as BL21 (DE3)) to screen and obtain positive Escherichia coli genetically engineered bacteria.

[0079] In some embodiments, the large-scale biofermentation step comprises: adding the positive genetically engineered Escherichia coli obtained by screening into a shake flask containing an antibiotic stock solution, and culturing on a shaking platform; preferably, the shaking platform culture conditions are 220 rpm and a constant temperature of 37°C.

[0080] In some embodiments, the steps of inducing protein expression include: (1) cooling the shake flask after culture to 16-30°C; (2) adding IPTG mother solution to induce expression; (3) placing the bacterial solution after induced expression in a centrifuge bottle, centrifuging at 6000 rpm and 4°C for 12 min, and then collecting the bacteria.

[0081] In some embodiments, the steps of purifying humanized type VIII collagen and optionally enzymatically cleaving the humanized type VIII collagen include: (1) crudely purifying humanized type VIII collagen on a Ni affinity chromatography column; (2) adding TEV enzyme for enzymatic cleavage in a certain proportion; and (3) finely purifying humanized type VIII collagen on an ion exchange column.

[0082] Example

[0083] The following examples are provided to illustrate the present invention. It should be understood by those skilled in the art that the examples are merely illustrative and non-limiting. The present invention is limited only by the scope of the appended claims.

[0084] Example 1: Construction and expression of recombinant humanized type VIII collagen

[0085] 1.1 Design of recombinant humanized type VIII collagen

[0086] A large-scale functional region screening of native human type VIII collagen (GenBank: CAA40748.1) revealed the following distinct functional regions.

[0087] GLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQGLQGPPGIPGIGGPSGPIGPPGIPGPKGEPGLP (SEQ ID NO.2)

[0088] GKPGFPGPKGDRGMGGVPGALGPRGEKGPIGAPGIGGPPGEPGLPGIPGPMGPPGAIGFPGPKGEGGIVGPQGPPGPKGEPGLQGFPGKPGFLGEVGPPGMRGLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQ (SEQID NO.5)

[0089] GVPGPPGFQGEPGPQGEPGPPGDR (SEQ ID NO.8)

[0090] To ensure the purification and stability of recombinant humanized type VIII collagen, the amino acid fragments of these regions were optimized by repeating n times and directly connecting them to obtain recombinant collagens VIII-1, VIII-2, and VIII-3; the corresponding amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7, respectively.

[0091] (1) Amino acid sequence of collagen VIII-1:

[0092] GLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQGLQGPPGIPGIGGPSGPIGPPGIPGPKGEPGLPGLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQGLQGPPGIPGIGGPSGPIGPPGIPGPKGEPGLPGLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQGLQGPPGIPGIGGPSGPIGPPGIPGPKGEPGLPGLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQGLQGPPGIPGIGGPSGPIGPPGIPGPKGEPGLP (SEQ ID NO.1);

[0093] (2) Amino acid sequence of collagen VIII-2:

[0094] GKPGFPGPKGDRGMGGVPGALGPRGEKGPIGAPGIGGPPGEPGLPGIPGPMGPPGAIGFPGPKGEGGIVGPQGPPGPKGEPGLQGFPGKPGFLGEVGPPGMRGLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQGKPGFPGPKGDRGMGGVPGALGPRGEKGPIGAPGIGGPPGEPGLPGIPGPMGPPGAIGFPGPKGEGGIVGPQGPPGPKGEPGLQGFPGKPGFLGEVGPPGMRGLPGPIGPKGEAGQKGVPGLPGVPGLLGPKGEPGIPGDQ (SEQ ID NO.4);

[0095] (3) Amino acid sequence of collagen VIII-3:

[0096] GVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGV PGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDRGVPGPPGFQGEPGPQGEPGPPGDR (SEQ IDNO.7).

[0097] Codon optimization was performed according to the E. coli expression system to obtain the nucleic acid sequences encoding collagen VIII-1, collagen VIII-2, and collagen VIII-3, as shown in SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9, respectively.

[0098] Collagen VIII-1 nucleic acid sequence:

[0099] GGGCTACCCGGACCGATCGGCCCGAAAGGTGAAGCGGGTCAGAAAGGTGTTCCGGGTCTGCCGGGTGTACCGGGCTTACTGGGTCCGAAGGGCGAGCCGGGCATTCCGGGGGATCAGGGTCTGCAAGGTCCTCCGGGCATCCCGGGGATCGGTGGTCCAAGCGGTCCTATCGGCCCGCCTGGTATCCCGGGCCCGAAAGGTGAACCGGGCCTGCCGGGCCTGCCGGGCCCGATTGGTCCGAAAGGTGAGGCTGGTCAGAAAGGTGTGCCGGGTCTGCCGGGCGTGCCCGGTTTGCTGGGTCCGAAGGGTGAGCCGGGCATTCCGGGCGACCAGGGCCTGCAGGGTCCACCGGGCATCCCGGGTATTGGTGGTCCATCTGGTCCGATCGGTCCGCCAGGCATCCCGGGCCCGAAGGGTGAACCGGGCCTGCCGGGTCTCCCAGGACCTATTGGTCCGAAGGGTGAGGCAGGCCAGAAAGGCGTTCCGGGTCTGCCGGGTGTTCCGGGTTTGCTGGGCCCGAAGGGTGAACCGGGCATCCCGGGCGATCAAGGATTGCAAGGTCCGCCTGGTATTCCGGGCATCGGCGGTCCGAGCGGTCCGATTGGTCCGCCAGGTATCCCGGGCCCGAAAGGTGAACCGGGTTTACCGGGGTTGCCGGGTCCGATTGGCCCGAAGGGTGAAGCGGGTCAGAAAGGTGTCCCGGGCCTGCCGGGCGTGCCGGGCTTGCTGGGTCCGAAGGGCGAGCCGGGTATCCCCGGTGACCAAGGCCTTCAAGGTCCGCCCGGCATCCCGGGCATTGGCGGTCCGTCCGGCCCGATTGGCCCGCCGGGCATTCCGGGTCCAAAGGGCGAGCCGGGCCTGCCG (SEQ ID NO.3)

[0100] Collagen VIII-2 nucleic acid sequence:

[0101] GGGAAACCCGGATTCCCGGGTCCAAAGGGTGACCGCGGTATGGGTGGCGTGCCGGGTGCGCTGGGTCCGCGTGGTGAGAAAGGTCCGATTGGCGCTCCGGGCATCGGCGGCCCACCGGGTGAACCTGGATTGCCGGGCATCCCGGGTCCGATGGGTCCGCCGGGAGCCATTGGTTTTCCGGGTCCGAAGGGTGAAGGTGGTATCGTGGGCCCACAGGGTCCGCCTGGGCCGAAGGGCGAACCGGGCCTGCAGGGTTTTCCGGGTAAACCGGGCTTTCTGGGTGAGGTTGGTCCGCCGGGCATGCGTGGCCTGCCGGGCCCGATCGGTCCGAAGGGCGAAGCAGGTCAGAAAGGCGTCCCGGGTCTGCCGGGCGTGCCGGGCTTGCTGGGCCCAAAAGGTGAGCCGGGTATTCCGGGGGATCAGGGTAAACCGGGCTTCCCGGGTCCGAAGGGTGACCGTGGTATGGGCGGTGTGCCGGGCGCGCTGGGTCCGCGTGGTGAAAAAGGCCCCATCGGCGCGCCAGGCATCGGCGGCCCGCCGGGCGAGCCGGGCTTACCGGGTATCCCGGGCCCGATGGGTCCGCCGGGTGCGATTGGTTTCCCGGGCCCAAAGGGCGAGGGTGGTATTGTTGGTCCACAGGGCCCACCGGGCCCCAAGGGTGAACCGGGCCTGCAAGGTTTTCCGGGGAAACCGGGCTTCCTCGGTGAAGTTGGTCCGCCGGGTATGCGCGGTCTGCCTGGCCCGATTGGCCCAAAGGGTGAGGCTGGCCAAAAAGGTGTTCCGGGCCTTCCGGGCGTCCCTGGTTTGCTGGGTCCGAAGGGTGAGCCGGGTATCCCGGGTGATCAA (SEQ ID NO.6)

[0102] Collagen VIII-3 nucleic acid sequence:

[0103] GGAGTACCCGGGCCACCGGGCTTCCAGGGCGAGCCTGGCCCTCAGGGTGAGCCGGGCCCGCCGGGTGATCGCGGTGTCCCGGGCCCACCGGGGTTCCAGGGTGAGCCGGGTCCACAGGGTGAACCGGGCCCACCGGGCGATAGAGGCGTGCCTGGCCCACCGGGTTTCCAAGGTGAGCCCGGTCCGCAGGGCGAGCCGGGCCCGCCGGGTGATCGTGGTGTTCCGGGCCCGCCGGGTTTTCAGGGCGAACCGGGTCCGCAAGGCGAACCGGGCCCGCCAGGCGACCGTGGTGTTCCGGGTCCGCCAGGCTTCCAGGGCGAGCCGGGCCCTCAAGGTGAGCCGGGTCCGCCGGGCGATCGTGGTGTCCCGGGTCCGCCGGGTTTCCAAGGCGAGCCGGGTCCGCAAGGTGAGCCGGGCCCTCCGGGTGATCGTGGTGTTCCGGGTCCTCCGGGTTTTCAAGGTGAACCGGGCCCGCAGGGTGAGCCGGGTCCGCCGGGTGACCGCGGTGTTCCGGGACCGCCGGGCTTTCAGGGTGAACCGGGCCCGCAAGGTGAACCGGGTCCGCCGGGCGACCGCGGTGTGCCGGGCCCGCCGGGCTTTCAAGGTGAACCCGGTCCGCAGGGCGAACCGGGCCCACCGGGCGACCGCGGCGTGCCGGGTCCGCCGGGCTTTCAGGGTGAGCCGGGTCCGCAGGGTGAGCCGGGTCCACCGGGTGACCGTGGTGTGCCGGGTCCGCCGGGATTCCAGGGTGAGCCGGGCCCACAAGGTGAACCGGGCCCCCCGGGCGACCGTGGTGTACCGGGACCGCCGGGTTTCCAGGGCGAACCGGGCCCGCAAGGCGAACCGGGCCCGCCGGGCGATCGT (SEQ ID NO.9)

[0104] The synthesized gene functional region was inserted into the pET-28a-Trx-His expression vector to obtain the corresponding recombinant expression plasmid.

[0105] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3). The specific process is as follows: (1) Take out the E. coli competent cells BL21 (DE3) from the ultra-low temperature refrigerator and place them on ice. When they are half melted, take 2 μl of the plasmid to be transformed and add it to the E. coli competent cells BL21 (DE3), and mix it slightly 2-3 times. (2) Place the mixture on ice for 30 minutes, then heat shock it in a 42°C water bath for 45-90 seconds, take it out and place it on ice for 2 minutes. (3) Transfer it to a biosafety cabinet and add 700 μl of liquid LB culture medium, then incubate it at 37°C and 220 rpm for 60 minutes. (4) Take 200 μl of the bacterial solution and evenly spread it on an LB plate containing kanamycin sulfate. (5) Incubate the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0106] Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing an antibiotic stock solution (ampicillin 100 mg / L). Incubate the culture in a shaker at 220 rpm and 37°C for a period of time until a haze appears. Cool the shake flask to 16-30°C and add IPTG (0.5 mM) to induce expression for a period of time. Aliquot the culture into centrifuge bottles and centrifuge at 6000 rpm and 4°C for 12 minutes. Collect the cells, record their weight, and remove samples for electrophoresis.

[0107] Resuspend the collected cells in a balanced working solution (200mM NaCl, 25mM Tris, 20mM imidazole), cool the solution to ≤15°C, homogenize twice or disrupt the cells by sonication, and collect the solution. Aliquot the disrupted solution into centrifuge bottles and centrifuge at 17,000 rpm and 4°C for 30 min. Collect the supernatant.

[0108] Recombinant humanized type VIII collagen was purified and enzymatically digested. The specific process was as follows: (1) Crude purification: a. Equilibration of the column: Use equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) to equilibrate the column at a flow rate of 10 mL / min. b. Sample loading: Add the supernatant after centrifugation to the column until the liquid has flowed away, at a flow rate of 5 mL / min. c. Washing of impurities: Add 100 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid has flowed away, at a flow rate of 10 mL / min. d. Collect the target protein: add 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) at a flow rate of 10 mL / min, collect the flow-through, and use UV-visible spectrophotometry to determine the protein concentration. Calculate the protein concentration according to the following formula (C (mg / ml) = A280 × dilution factor × extinction coefficient), and perform electrophoresis detection. e. Wash the column with 1 M imidazole working solution at a flow rate of 10 mL / min. (2) Enzyme digestion: Add TEV enzyme at a ratio of 20:1 between the total amount of protein and the total amount of TEV enzyme, and digest at 16°C for 2 h. Place the protein solution after digestion in a dialysis bag and dialyze at 4°C for 2 h. Then transfer it to a new dialysis solution (20 mM sodium chloride, 20 mM Tris) and dialyze it overnight at 4°C. (3) Purification: a. Equilibrate the column: Use Solution A (20 mM Tris, 20 mM NaCl) to equilibrate the column at a flow rate of 10 ml / min. b. Load the sample: Load the sample at a flow rate of 5 ml / min, collect the flow-through sample, and perform electrophoresis. Store the protein at 4°C. c. Elute: Wash the column for 5 CV with Solution B (1 M NaCl, 20 mM Tris). d. Wash the column.

[0109] The purified recombinant protein was detected by SDS-PAGE. The electrophoresis results were as follows: Figure 1-Figure 3 After purification, the protein yield of VIII-1 was 1.29%. The target protein was abundant and of high purity. The theoretical molecular weight after purification was 25.9KD. The apparent molecular weight after purification was consistent with the theoretical molecular weight ( Figure 1 VIII-2 The target protein has many impurities. After purification, the theoretical molecular weight is 25.98KD. The apparent molecular weight after purification is consistent with the theoretical molecular weight ( Figure 2 The purification yield of VIII-3 was 0.35%, the amount of protein was small, the theoretical molecular weight after purification was 27.2KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight ( Figure 3 ).

[0110] Example 2: UV-scanning analysis of recombinant humanized type VIII collagen by circular dichroism

[0111] Experimental methods

[0112] (1) Instrument parameter setting

[0113] Bandwidth: 1.0nm

[0114] Step: 1.0nm

[0115] Measuring range: 190-260nm (far-UV scanning) / 250-340nm (near-UV scanning)

[0116] Time per point: 0.5s

[0117] Repeat 3 times

[0118] Cell Length: 10mm|0.5mm

[0119] Temperature Room temperature

[0120] (2) Far and near UV scanning of standard products

[0121] The scanning wavelength was set to 180-340 nm for background test and blank buffer test, and then the near- and far-UV circular dichroism absorption of 1 mg / mL CSA standard solution in the range of 180-340 nm was collected.

[0122] Sample processing

[0123] Protein samples were collected and concentrated using 10KD ultrafiltration concentrators (Millipore) to a protein concentration of 1 mg / ml.

[0124] (4) Far UV scanning of samples

[0125] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry, collect the background first, then collect the blank buffer, then add an appropriate amount of test sample to the cuvette, perform far-UV scanning at 190-260nm according to the above parameters and collect data.

[0126] (5) Sample near-UV scanning

[0127] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry it. Collect the background first, then collect the blank buffer solution, then add an appropriate amount of test sample to the cuvette, perform near-UV scanning at 250-340nm according to the above parameters and collect data.

[0128] (6) Scanning spectrum processing

[0129] All scanned images were processed by subtracting baseline and smoothing using the software Pro-Data Viewer.

[0130] The circular dichroism UV scanning analysis results of recombinant collagen VIII-1, VIII-2, and VIII-3 are as follows: Figure 4-6 The expressed recombinant humanized type VIII collagen had positive peaks at 220-230 nm, indicating that collagen VIII-1, VIII-2, and VIII-3 all had triple helical structures.

[0131] Example 3: Bioactivity Detection of Recombinant Humanized Type VIII Collagen

[0132] The method for detecting collagen activity can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0133] (1) The concentration of the protein samples to be tested was detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Institute, No.: 380002) and the recombinant proteins VIII-1, VIII-2, and VIII-3 provided by the present invention. Specifically, 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). Note that the test 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, it is not affected by the content of chromophores, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.). After the protein concentration was detected, the concentration of all the proteins to be tested was adjusted to 0.5 mg / mL with PBS.

[0134] (2) Sample preparation: Use the sample stock solution directly for the experiment; dilute the positive control bovine type I collagen (PC) to 1 mg / ml using D-PBS; the negative control is D-PBS buffer (NC).

[0135] (3) Coating: Collagen at different concentrations (0.5, 1, 1.5 mg / ml) and positive and negative controls were added to the ELISA plate, 100 μL per well, 5 replicate wells were set up for each group, and the plates were incubated at 4°C overnight.

[0136] (4) Blocking: Discard the supernatant, add 100 μL of 1% BSA (heat-inactivated at 56°C for 30 min), and incubate at 37°C for 60 min. Discard the supernatant and wash three times with D-PBS solution.

[0137] (5) Cell inoculation: Add 10 5 Resuspend well-cultured 3T3 / NIH cells in D-PBS and incubate at 37°C for 120 min. Wash each well three times with D-PBS solution.

[0138] (6) Detection: The absorbance at OD450nm was measured using the CCK8 detection kit (manufacturer: Beyotime, catalog number C0038). The degree of cell adhesion was calculated according to the following formula. The cell adhesion rate reflects the cell adhesion ability of collagen. The higher the cell adhesion ability, the better the external environment provided to the cells in a short time, helping the cells to adhere.

[0139] Relative cell adhesion = {(OD1-OD0) / (OD2-OD0)}×100%,

[0140] Where: OD1: average UV absorbance of each replicate well of the test collagen sample at 450 nm; OD2: average UV absorbance of each replicate well of the control collagen sample at 450 nm; OD0: average UV absorbance of each replicate well of the blank control group at 450 nm.

[0141] (7) Statistical analysis: Two-tailed t-test was used to analyze the statistical differences between the target recombinant humanized collagen and the negative control, where *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 7 As shown in the figure, compared with the D-PBS group, the positive control group had a significant effect on promoting cell adhesion. Recombinant humanized collagen VIII-1, collagen VIII-2, and collagen VIII-3 also promoted cell adhesion, and the effects were significantly higher than the positive control group.

Claims

1. Recombinant humanized type VIII collagen, characterized in that: The invention comprises n repeating units, wherein the repeating units are connected directly or through a linker, and the repeating units comprise the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:8 or a variant thereof, wherein the variant is (1) an amino acid sequence obtained by substituting, deleting or inserting one or more amino acids on the basis of SEQ ID NO:2 or SEQ ID NO:8; or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:2 or SEQ ID NO:8; Preferably, n is an integer of 1-20; Preferably, n is 4, 8 or 12; Preferably, the repeating units are directly connected; Preferably, the collagen comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.7, or an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:7, or an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids on the basis of SEQ ID NO:1 or SEQ ID NO:

7.

2. A fusion protein comprising the recombinant humanized type VIII collagen according to claim 1 and a purification tag and / or a leader; preferably, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag.

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

4. A vector comprising the polynucleotide of claim 3; preferably, the vector is an expression vector; preferably, the vector comprises a control element operably linked to the polynucleotide; preferably, the control element is selected from a promoter, a terminator and / or an enhancer.

5. A host cell comprising the polynucleotide of claim 3, or the vector of claim 4; preferably, the host cell is a bacterium, a fungus or an animal cell; preferably, the bacterium comprises Escherichia coli; preferably, the fungus comprises yeast, such as Saccharomyces cerevisiae. A collagen trimer comprising the recombinant type VIII humanized collagen according to claim 1.

7. A method for producing the recombinant humanized type VIII collagen according to claim 1 or the fusion protein according to claim 2, characterized in that: The steps include: (1) culturing the host cell according to claim 5 under suitable culture conditions; (2) harvesting host cells and / or culture medium containing recombinant type VIII humanized collagen or fusion protein; and optionally (3) Purifying the recombinant humanized type VIII collagen or fusion protein.

8. A composition comprising one or more of the recombinant humanized type VIII collagen according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5 and the collagen trimer according to claim 6.

9. The composition according to claim 8, wherein the composition is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives; preferably, the composition is a surface composition, an injectable composition or an oral composition; preferably, the composition is a composition in the form of a solution, a lyophilized powder, a gel, a sponge or a fiber.

10. Use of the recombinant humanized type VIII collagen according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5 and the collagen trimer according to claim 6 in the preparation of one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone / cartilage regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

11. A method for promoting cell adhesion, comprising the step of contacting the recombinant humanized type VIII collagen according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5, the composition according to claims 7-8 and / or the collagen trimer according to claim 6 with cells; preferably, the cells are animal cells; preferably, the animal cells are mammalian cells; preferably, the mammalian cells are human cells; preferably, the promoting of cell adhesion is performed in vitro.

12. A method for performing plastic surgery, tissue injection filling, ophthalmic treatment, nerve repair or vascular repair on a subject in need thereof, comprising administering to the subject the recombinant humanized type VIII collagen according to claim 1 and / or the collagen trimer according to claim 6; preferably, the administration is oral administration or injection administration; preferably, the subject is a human.

13. Use of the recombinant humanized type VIII collagen according to claim 1 and / or the collagen trimer according to claim 6 in the preparation of a medicament for a disease or condition associated with type VIII collagen deficiency; preferably, the disease or condition is anterior segment dysplasia.

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