Recombinant humanized collagen type viii and uses thereof

Abstract

The present disclosure provides a recombinant humanized collagen VIII, which is a triple helix structure, has good cell adhesion activity, and is applied to human body without immune rejection and allergic reaction.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology, and specifically relates to human structural materials and their biosynthetic preparation methods. Background Technology

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

[0003] Type VIII collagen, a member of the collagen family, is primarily secreted by endothelial cells, epithelial cells, and Schwann cells, and is distributed throughout ocular and vascular tissues. Type VIII collagen plays a crucial role in maintaining corneal transparency and normal structure. Interacting with other types of collagen and other components of the cornea, it forms an ordered fibrous network structure, ensuring a smooth and uniform corneal shape. This allows light to pass through the cornea smoothly into the eye, providing the foundation for clear vision. Type VIII collagen is also distributed in the lens of the eye, playing a vital role in maintaining its normal structure and transparency. It helps ensure proper refraction and transmission of light within the lens, maintaining good vision. In vascular tissues, it helps maintain the integrity and stability of the blood vessel walls. Distributed around vascular endothelial cells, it, along with other extracellular matrix components, provides physical support to the endothelial cells, enabling the blood vessels 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, removing 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 to ensure normal tissue growth and effective repair after injury. The interaction between type VIII collagen and cell surface receptors transmits specific signals, guiding cells to differentiate in specific directions. For example, during embryonic development, it can influence the differentiation of endothelial cells into different types of vascular cells, participating in the formation and development of the vascular system. Type VIII collagen can also serve as a temporary extracellular matrix, providing a scaffold for cells migrating to the wound site to attach and migrate. It attracts various cells, including fibroblasts and endothelial cells, to the wound site, where they migrate, proliferate, and begin to synthesize and secrete new extracellular matrix components, laying the foundation for wound healing.

[0005] To date, most collagen used in various studies has been derived from animal tissues and skin extracts. However, collagen extracted from animals has poor water solubility and weak processability, directly limiting the development of many potential applications. The production of full-length type VIII collagen using genetic engineering also presents several problems: for example, there are differences in codon usage frequencies between prokaryotic and eukaryotic cells. The full-length type VIII collagen gene originates from eukaryotes, and its codon usage may differ from the preferences of the prokaryotic host. This can cause ribosomes to pause at rare codons during translation in prokaryotic cells, reducing protein synthesis efficiency and even affecting the correct folding and elongation of nascent peptide chains. Furthermore, when using eukaryotic systems for production, excessive accumulation of recombinant proteins within cells may negatively impact cellularity, potentially leading to lower-than-expected expression levels of full-length type VIII collagen. Additionally, yeast cells may have limited secretory capacity for some high-molecular-weight proteins, resulting in some proteins not being effectively secreted extracellularly. Summary of the Invention

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

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

[0008] In some embodiments, the linker comprises one or more 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 obtained by substitution, deletion, or insertion of one or more amino acids based on 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 with 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 contains 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 having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1 or SEQ ID NO:7.

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

[0013] A second aspect of the present invention provides a fusion protein comprising the recombinant type VIII humanized collagen described in the first aspect of the present invention and a purified tag and / or precursor.

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

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

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

[0018] In some embodiments, the vector is an expression vector; in some embodiments, the vector includes a control element operatively linked to a polynucleotide; in some embodiments, the control element is selected from promoters, terminators, and / or enhancers.

[0019] The fifth aspect of the present invention provides a host cell comprising the polynucleotides described in the third aspect of the present invention, or the vectors described in the fourth aspect of the present invention.

[0020] In some embodiments, the host cell is a bacterium, fungus, 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 type VIII humanized 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 type VIII humanized 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) Culture the host cells described in the fifth aspect of the present invention under suitable culture conditions;

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

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

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

[0027] In some embodiments, the composition is one or more of the following: bio-dressings, biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives; preferably, the composition is a surface composition, an injectable composition or an oral composition; preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge or fiber.

[0028] The ninth aspect of this invention provides the use of the recombinant type VIII humanized collagen described in the first aspect of this invention, the fusion protein described in the second aspect of this invention, the polynucleotide described in the third aspect of this invention, the carrier described in the fourth aspect of this invention, the host cell described in the fifth aspect of this invention, or the collagen trimer described in the sixth aspect of this invention in the preparation of one or more of the following: biological 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, 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, comprising the step of contacting the recombinant type VIII humanized collagen of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the polynucleotide of the third aspect of the present invention, the carrier of the fourth aspect of the present invention, the host cell of the fifth aspect of the present invention, the collagen trimer of the sixth aspect of the present invention, and / or the composition of the eighth aspect of the present invention with the cell.

[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 cell adhesion promotion is carried out in vitro.

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

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

[0033] The advantages of this invention include:

[0034] 1. The recombinant type VIII collagen of the present invention is derived from natural human type VIII collagen and will not produce 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, has a large expression level and is suitable for subsequent purification, has high production efficiency and no pollution, and has good prospects for industrial production.

[0036] 3. The recombinant type VIII collagen of the present invention has higher cell adhesion activity compared with the control group, and has been verified by circular dichroism spectroscopy to have a stable triple helix structure and good bioactivity. 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 diseases, ophthalmic diseases, dermatology and cosmetic medicine, nervous system repair and degenerative diseases and tissue engineering. Attached Figure Description

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

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

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

[0040] Figure 4 This is a CD diagram of collagen VIII-1.

[0041] Figure 5 This is a CD diagram of collagen VIII-2.

[0042] Figure 6 This is a CD diagram of collagen VIII-3.

[0043] Figure 7 The attached diagram shows the cell adhesion of three recombinant type VIII humanized collagens. 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] Terminology Definition

[0046] As used in this article, "recombinant collagen" is a new type of biomaterial that uses cutting-edge structural biology, genetic engineering and other technologies to screen and prepare a gene encoding the functional region of human collagen specific type as a template, and obtains an amino acid sequence that is the same as or similar to that of human collagen.

[0047] As used herein, “recombinant humanized type VIII collagen” refers to a recombinant protein consisting of or substantially consisting of sequences derived from human type VIII collagen. In this context, recombinant humanized type VIII collagen may consist of or substantially consist of fragments or multiple repeats of fragments 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., substitution, deletion, insertion, or addition), “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 peptide production, 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 enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may contain the nucleic acids of this invention for introduction into cells for expression. Vectors may contain expression control elements operatively linked to the nucleic acids, such as promoters, terminators, and / or enhancers.

[0051] As used herein, the term "expression vector" refers to a straight or circular DNA molecule containing a polynucleotide encoding a polypeptide and operatively linked to a control sequence provided for its expression. In this paper, the expression vector is an *E. coli* expression vector.

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

[0053] As used herein, the term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding region of the polynucleotide encoding the polypeptide.

[0054] As used herein, the term "host cell" refers to a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques. Host cells can be eukaryotic or prokaryotic cells. For example, eukaryotic cells include yeast cells, animal cells, and / or insect cells. Prokaryotic cells can be E. coli cells.

[0055] Recombinant Type VIII humanized collagen

[0056] This invention provides recombinant type VIII humanized collagen. The recombinant type VIII humanized collagen of this invention may comprise one or more repeating units, each repeating unit containing 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 mutating (substituting, adding, or deleting) 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 having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2 or SEQ ID NO:8.

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

[0060] In some implementations, 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 implementations, the number of repeating units is 4, 8, or 12.

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

[0063] In some implementations, the repeating units are directly connected.

[0064] In some implementations, one or more amino acid residues are spaced between each repeating unit.

[0065] In some implementations, when a mutation exists in the repeating unit, the resulting recombinant collagen retains comparable or superior cell adhesion compared to collagen obtained from an unmutated repeating unit.

[0066] In some embodiments, the recombinant type VIII humanized collagen of the present invention is synthetic 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 having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1 or SEQ ID NO:7, or 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.

[0068] Recombinant type VIII humanized collagen encodes nucleic acid

[0069] This 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 expressed.

[0071] In some implementations, the polynucleotide encoding recombinant type VIII humanized collagen can be operatively linked to expression control elements, such as promoters, terminators, and / or enhancers, to form a nucleic acid or expression cassette.

[0072] In some embodiments, the polynucleotide encoding recombinant type VIII humanized collagen may also include nucleotides encoding purification tags, such as His tags, GST tags, MBP tags, SUMO tags, or NusA tags, or nucleotides encoding leader sequences to facilitate peptide purification or secretion.

[0073] Compositions and their applications

[0074] This invention also provides compositions comprising recombinant type VIII humanized collagen. These compositions include, but are not limited to, bio-dressings, biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological 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.

[0075] The composition can be applied by means including, but not limited to, injection, topical application, or oral administration.

[0076] Production of recombinant type VIII humanized collagen

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

[0078] 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 (such as PET-28a-Trx-His) to obtain a recombinant expression plasmid; (3) transforming the recombinant expression plasmid into competent Escherichia coli cells (such as BL21(DE3)) and screening to obtain positive Escherichia coli genetically engineered bacteria.

[0079] 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 on a shaker; preferably, the shaker culture conditions are 220 rpm and 37°C constant temperature.

[0080] In some embodiments, the steps for 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.

[0081] In some embodiments, the purification and optional enzymatic digestion steps of type VIII humanized collagen include: (1) crude purification of type VIII humanized collagen on a Ni affinity chromatography column; (2) enzymatic digestion with TEV enzyme in a certain proportion; and (3) purification of type VIII humanized collagen on an ion exchange column.

[0082] Example

[0083] 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.

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

[0085] 1.1 Design of recombinant type VIII humanized collagen

[0086] Large-scale functional region screening of natural human type VIII collagen (GenBank: CAA40748.1) yielded the following different 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 type VIII humanized collagen, the amino acid fragments of these regions were optimized by repeated n times and directly linked to obtain recombinant collagen 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 IDNO.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 using the E. coli expression system to obtain the coding nucleic acid sequences for 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 was as follows: (1) The E. coli competent cells BL21(DE3) were taken out of the ultra-low temperature freezer and placed on ice. When they were half-thawed, 2 μl of the plasmid to be transformed was added to the E. coli competent cells BL21(DE3) and mixed slightly 2-3 times. (2) The mixture was placed on ice for 30 min, and then heat-shocked in a water bath at 42℃ for 45-90 s. After taking it out, it was placed on ice for 2 min. (3) It was transferred to a biosafety cabinet and 700 μl of liquid LB medium was added. Then it was cultured at 37℃ and 220 rpm for 60 min. (4) 200 μl of bacterial solution was evenly spread on LB plates containing kanamycin sulfate. (5) The plates were cultured in an incubator at 37℃ for 15-17 h until uniformly sized colonies grew.

[0106] Pick 5-6 single colonies from the transformed LB agar plates and place them in a shake flask containing antibiotic stock solution (ampicillin 100 mg / L). Incubate at 220 rpm and 37°C in a constant temperature shaker for a certain period of time until the colonies appear as a mist. Then, cool the shake flasks to 16-30°C, add IPTG (0.5 mM) to induce expression for a period of time, aliquot the bacterial culture into centrifuge bottles, centrifuge at 6000 rpm and 4°C for 12 min, collect the bacterial cells, record the cell weight, and perform electrophoresis analysis.

[0107] 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.

[0108] The recombinant type VIII humanized collagen was purified and enzymatically digested. The specific process was as follows: (1) Crude purification: a. Equilibrate the column: Equilibrate the column with equilibration buffer (200mM sodium chloride, 25mM Tris, 20mM imidazole) at a flow rate of 10mL / min. b. Load the sample: Add the supernatant after centrifugation to the column until the liquid is completely discharged, at a flow rate of 5mL / min. c. Wash away impurities: Add 100 mL of washing buffer (200 mM sodium chloride, 25 mM Tris, 20mM imidazole) until the liquid is completely discharged, at a flow rate of 10mL / min. d. Collect the target protein: Add 20 mL of elution buffer (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole), at a flow rate of 10 mL / min, and collect the flow-through. Detect the protein concentration using UV-Vis spectrophotometry. Calculate the protein concentration using the following formula (C (mg / ml) = A280 × dilution factor × extinction coefficient), and perform electrophoresis. e. Wash the column with 1 M imidazole working solution at a flow rate of 10 mL / min. (2) Enzyme digestion: Add TEV enzyme at a ratio of total protein to total TEV enzyme of 20:1, and digest at 16℃ for 2 h. Place the digested protein solution into a dialysis bag and dialyze at 4℃ for 2 h. Then transfer it to a new dialysis buffer (20 mM sodium chloride, 20 mM Tris) and dialyze overnight at 4℃. (3) Purification: a. Column equilibration: Equilibrate the column with solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 ml / min. b. Sample loading: Load the sample at a flow rate of 5 ml / min, collect the flow-through sample, and perform electrophoresis. Store the protein at 4°C. c. Elution: Wash the column with solution B (1 M sodium chloride, 20 mM Tris) for 5 CVs. d. Column washing.

[0109] SDS-PAGE analysis of the purified recombinant protein, electrophoresis results are as follows: Figures 1-3 As shown. After purification, the protein yield of VIII-1 was 1.29%. The amount of the target protein after purification was relatively large, and the purity was high. The theoretical molecular weight after purification was 25.9 KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight. Figure 1 The target protein VIII-2 had many impurities. After purification, its theoretical molecular weight was 25.98 KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight. Figure 2 The purification yield of VIII-3 was 0.35%, with a relatively small amount of protein. The theoretical molecular weight after purification was 27.2 kDa, and the apparent molecular weight after purification was consistent with the theoretical molecular weight. Figure 3 ).

[0110] Example 2: Circular dichroism UV-Vis analysis of recombinant type VIII humanized collagen

[0111] Experimental methods

[0112] (1) Instrument parameter settings

[0113] Bandwidth: 1.0nm

[0114] Step 1.0nm

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

[0116] Time per point: 0.5 seconds

[0117] Repeat 3 times

[0118] Cell Length: 10mm | 0.5mm

[0119] Temperature room temperature

[0120] (2) Near and far ultraviolet scanning of standard samples

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

[0122] Sample processing

[0123] Take protein samples and concentrate them to a protein concentration of 1 mg / ml using a 10 kDa ultrafiltration concentrator (Millibo).

[0124] (4) Far-ultraviolet scanning of samples

[0125] Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a far-ultraviolet scan at 190-260 nm according to the above parameters and collect the data.

[0126] (5) Near-ultraviolet scanning of samples

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

[0128] (6) Scanning image processing

[0129] All scanned spectra were processed using the software Pro-Data Viewer to extract baselines and smooth out the images.

[0130] The circular dichroism spectroscopy UV-Vis analysis results of recombinant collagen VIII-1, VIII-2, and VIII-3 are as follows: Figure 4-6 As shown, the expression of recombinant type VIII humanized collagen showed positive peaks at 220-230 nm, indicating that collagen VIII-1, VIII-2, and VIII-3 all have a triple helix structure.

[0131] Example 3: Bioactivity assay of recombinant type VIII humanized 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 using ultraviolet absorption, including bovine type I collagen (China National Institutes for Food and Drug Control, No.: 380002) and recombinant proteins VIII-1, VIII-2, and VIII-3 provided in this 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 detection must be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far-ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues that are not colorimetric by Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43-45.). After the protein concentration was measured, the concentration of all the proteins to be tested was adjusted to 0.5 mg / mL with PBS.

[0134] (2) Sample preparation: The original sample solution was used directly for the experiment; the positive control bovine type I collagen (PC) was diluted to 1 mg / ml with D-PBS for later use; the negative control was D-PBS buffer (NC).

[0135] (3) Coating: Add different concentrations (0.5, 1, 1.5 mg / ml) of collagen, positive control and negative control to the microplate, 100 μL per well, 5 replicates per group, and incubate overnight at 4°C.

[0136] (4) Blocking: Discard the supernatant, add 100 μL of 1% BSA (heat inactivated at 56℃ for 30 min), and incubate at 37℃ for 60 min. Discard the supernatant and wash 3 times with D-PBS solution.

[0137] (5) Cell seeding: Add 10g of the solution to each well. 5 3T3 / NIH cells in good condition, resuspended in D-PBS, were incubated at 37°C for 120 min. Each well was washed three times with D-PBS solution.

[0138] (6) Detection: The absorbance at OD450nm was measured using the CCK8 assay kit (manufacturer: Beyotime, product catalog number C0038). The cell adhesion degree 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 can be 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: the average UV absorbance of each duplicate well of the collagen sample at 450nm; OD2: the average UV absorbance of each duplicate well of the control collagen sample at 450nm; OD0: the average UV absorbance of each duplicate well of the blank control group at 450nm.

[0141] (7) Statistical Analysis: A two-tailed t-test was used to analyze the statistical differences between the target recombinant humanized collagen and the negative control. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results are as follows: Figure 7 As shown, compared with the D-PBS group, the positive control had a significant effect on promoting cell adhesion. Recombinant humanized collagen VIII-1, collagen VIII-2, and collagen VIII-3 also had a promoting effect on cell adhesion, and the effect was significantly higher than that of the positive control.

Claims

1. Recombinant type VIII humanized collagen, characterized in that, Composed of n directly linked repeating units, the amino acid sequence of which is either SEQ ID NO:2 or SEQ ID NO:8; where n is an integer from 4 to 12, the recombinant type VIII humanized collagen has cell adhesion-promoting activity.

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

3. The recombinant type VIII humanized collagen according to claim 1, wherein the amino acid sequence of the recombinant type VIII humanized collagen is SEQ ID NO.1 or SEQ ID NO.

7.

4. A fusion protein comprising any one of claims 1-3 recombinant type VIII humanized collagen and a purified tag.

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

6. A polynucleotide encoding the recombinant type VIII humanized collagen of any one of claims 1-3 or the fusion protein of claim 4 or 5.

7. The polynucleotide according to claim 6, comprising the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:9 or a degenerate sequence thereof.

8. A vector comprising the polynucleotide of claim 6 or 7.

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

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

11. The carrier according to claim 10, wherein the control element is selected from promoters, terminators, and / or enhancers.

12. A host cell comprising the polynucleotide of claim 6 or 7, or the vector of any one of claims 9-11.

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

14. The host cell according to claim 13, wherein the bacteria is Escherichia coli.

15. The host cell according to claim 13, wherein the fungus is yeast.

16. The host cell according to claim 15, wherein the yeast is Saccharomyces cerevisiae.

17. Collagen trimer, wherein the collagen is recombinant humanized type VIII collagen as described in any one of claims 1-3.

18. A method for producing recombinant type VIII humanized collagen according to any one of claims 1-3 or the fusion protein according to claim 4 or 5, characterized in that, Includes the following steps: (1) Culture the host cell according to any one of claims 12-16 under suitable culture conditions; (2) Harvesting host cells and / or culture medium containing recombinant type VIII humanized collagen or fusion protein; and optionally (3) Purify the recombinant type VIII humanized collagen or fusion protein.

19. A composition comprising one or more of the following: recombinant humanized type VIII collagen according to any one of claims 1-3, the fusion protein according to claim 4 or 5, and the collagen trimer according to claim 17.

20. The composition according to claim 19, wherein the composition is one or more of the following: human biomimetic materials, organoid culture materials, coating materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives.

21. The composition of claim 20, wherein the composition is a surface composition, an injectable composition, or an oral composition.

22. The composition according to claim 20 or 21, wherein the composition is in the form of a solution, lyophilized powder, gel, sponge or fiber.

23. The composition according to claim 20, wherein the biomimetic material is one or more of the following: bio-dressing, cardiovascular stent material, plastic and cosmetic material, ophthalmic material, obstetric and gynecological biomaterial, nerve repair and regeneration material, liver tissue material, and vascular repair and regeneration material.

24. The composition of claim 23, wherein the cosmetic material is a tissue injection filler.

25. The use of recombinant type VIII humanized collagen according to any one of claims 1-3, the fusion protein according to claim 4 or 5, the polynucleotide according to claim 6 or 7, the carrier according to any one of claims 8-11, the host cell according to any one of claims 12-16, or the collagen trimer according to claim 17 in the preparation of one or more of the following: human biomimetic materials, organoid culture materials, coating materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives.

26. The use according to claim 25, wherein the biomimetic material is one or more of the following: biological dressings, cardiovascular stent materials, plastic and cosmetic materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone regeneration materials, liver tissue materials, and vascular repair and regeneration materials.

27. The use according to claim 26, wherein the cosmetic material is a tissue injection filler.

28. The use according to claim 25, wherein the biomimetic material is a cartilage regeneration material.

29. An in vitro method for promoting cell adhesion, comprising the step of contacting cells with recombinant humanized type VIII collagen according to any one of claims 1-3, the fusion protein according to claim 4 or 5, the composition according to claim 19, and / or the collagen trimer according to claim 17.

30. The in vitro method according to claim 29, wherein the cell is an animal cell.

31. The in vitro method according to claim 30, wherein the animal cell is a mammalian cell.

32. The in vitro method according to claim 31, wherein the mammalian cell is a human cell.

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