Preparation method for biosynthetic human structural material type iv collagen

Abstract

Provided is a method for biosynthesis of a human structural material type IV collagen. Provided herein is a recombinant collagen, comprising sequences as shown in SEQ ID NO: 1 and the like. The recombinant collagen herein has good cell adhesion activity. The method herein relates to using the genetic engineering technology to produce the recombinant collagen, and overcomes defects in the prior art.

Description

Preparation method of biosynthetic human structural material type IV collagen

[0001] This application claims priority to the Chinese invention patent application with application date of October 25, 2023, application number 202311391711.1, and invention name “Method for preparing type IV collagen, a biosynthetic human structural material”. Technical Field

[0002] The present invention belongs to the technical field of biomedicine and relates to a recombinant humanized type IV collagen and a preparation method and application thereof. Background Art

[0003] Collagen (Collagen, COL), also known as collagen for short, is a helical, fibrous functional protein composed of three peptide chains. It is also the main component of the extracellular matrix, with abundant content and wide coverage. The content of collagen in the human body accounts for 25%-30% of the total protein. It is mainly found in the skin, tendons and bones. It plays an important role in protecting and connecting various tissues and plays an important physiological function in the body. Type IV collagen is the main protein that constitutes the basement membrane together with laminin. It uses a trimer with a helical structure composed of three α chains as a structural unit. Although the subunits that make up the trimer are different depending on the tissue, in many tissues, such as the liver, the trimer is composed of two α1 chains and one α2 chain. The type IV collagen molecule has a structure unique to the type IV collagen molecule at both ends of the TH domain that forms the helical structure. The structure at the N-terminus is called the 7S domain, and the structure at the C-terminus is called the NC1 domain.

[0004] Collagen has good biocompatibility, degradability and low antigenicity. Its unique biological structure has made it a research hotspot in recent years. It can be widely used in many fields such as biomedicine, cosmetics, health products, and food.

[0005] The human body contains 28 different types of collagen, which can be divided into two categories based on their fibrillar structure: fibrous and non-fibrous. Fibrous collagen primarily serves as a scaffold, anchoring cells and providing tensile strength and rigidity to tissues. Non-fibrous collagen is further subdivided into basal collagen, short-chain collagen, and transmembrane collagen, each with distinct functions.

[0006] Traditional collagen production methods involve purifying animal tissues using acid, alkali, and enzymatic hydrolysis to extract collagen derivatives. However, the collagen obtained through these methods has lost its original biological activity and is poorly water-soluble, poorly binds to the human body, and poses risks of viral infection and allergenicity, rendering it unable to fully function. Some research institutions have also attempted to express human collagen in vitro through conventional recombinant expression methods, but these methods are costly, require lengthy production cycles, and are unsuitable for large-scale production.

[0007] Therefore, the market is in urgent need of a collagen material that has excellent biomaterial properties, an amino acid sequence that is highly homologous to that of the human body, and can be prepared in large quantities in an industrial system.

[0008] Summary of the Invention

[0009] The inventors conducted a large-scale functional region screening of human type IV collagen and discovered 11 recombinant collagen proteins. These recombinant collagen proteins can be expressed in Escherichia coli and purified. Furthermore, the inventors found that these recombinant collagen proteins had high yields, high purity after purification, and exhibited higher activity than the positive control (bovine type I collagen) in cell adhesion assays.

[0010] In one aspect, the present invention provides a recombinant collagen comprising one or more repeating units, which are connected directly or through a linker, and the repeating unit comprises an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1 (Gakgdkgskgevgfpglagspgipgskgeq) or 28 (Gptgpagqkgepgsdgipgsagekgepglp), wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence 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% or 100% identity with the amino acid sequence.

[0011] In one embodiment, the plurality of repeating units is 2-50 repeating units, such as 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 4-10 or 6-10 repeating units. For example, the number of repeating units can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50 or a range therebetween.

[0012] In one embodiment, the linker comprises one or more amino acid residues, e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0013] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion.

[0014] In one embodiment, the substitutions are conservative amino acid substitutions.

[0015] In one embodiment, the recombinant collagen is recombinant human type IV collagen or recombinant type IV humanized collagen.

[0016] In one embodiment, the recombinant collagen has cell adhesion activity.

[0017] In one embodiment, the variant of SEQ ID NO: 1 comprises the following mutations: addition of Gfpgfp (SEQ ID NO: 34) or an N-terminal truncated fragment of 1-5 amino acid residues in length of SEQ ID NO: 34 to the N-terminus of the amino acid sequence of SEQ ID NO: 1, and / or addition of GFMGPPGPQGQPGLP (SEQ ID NO: 35) or a C-terminal truncated fragment of 1-14 amino acid residues in length of SEQ ID NO: 35 to the C-terminus of the amino acid sequence of SEQ ID NO: 1, or truncation of 1-5 amino acid residues at the C-terminus of the amino acid sequence of SEQ ID NO: 1.

[0018] In one embodiment, the variant of SEQ ID NO: 28 comprises the following mutation: Glpgtp (SEQ ID NO: 36) or a 1-5 amino acid residue long N-terminal truncation thereof is added to the N-terminus of the amino acid sequence of SEQ ID NO: 28.

[0019] In one embodiment, a variant of SEQ ID NO: 1 is selected from SEQ ID NO: 4 (Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), 7 (GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP), 10 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), 13 (Gfpgfpgakgdkgskgevgfpglagsp gipgskgeqgfm), 16(Gfpgfpgakgdkgskgevgfpglagspgipgskgeq), 19(Gakgdkgskgevgfpglagspgipgskgeqgfm gppgpqgqpglp), 22 (Gakgdkgskgevgfpglagspgipgskgeqgfm), or 31 (Gfpgfpgakgdkgskgevgfpglagspgipgsk).

[0020] In one embodiment, the variant of SEQ ID NO: 28 is SEQ ID NO: 25 (Glpgtpgptgpagqkgepgsdgipgsagekgepglp).

[0021] In one embodiment, the recombinant collagen comprises an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence.

[0022] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion. In one embodiment, the substitution is a conservative amino acid substitution.

[0023] In another aspect, a nucleic acid is provided that encodes a recombinant collagen as described herein. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, the nucleotide sequence is a nucleotide sequence codon-optimized for expression in a eukaryotic host cell or a prokaryotic host cell, such as yeast or E. coli. In one embodiment, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33.

[0024] In another aspect, a vector is provided, which comprises a nucleic acid as described herein. In one embodiment, the vector comprises an expression control element, a purification tag, and / or a leader sequence nucleotide that is operably linked to the nucleic acid. In one embodiment, the expression control element is selected from a promoter, a terminator, or an enhancer. In one embodiment, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag. In one embodiment, the vector is an expression vector or a cloning vector, preferably pET-28a(+). pET-28a(+) may comprise N-terminal His, Thrombin, and T7 protein tags, and a C-terminal His tag. In this article, the N-terminus of the recombinant collagen may comprise an enzyme cleavage site for ease of purification.

[0025] In another aspect, a host cell is provided, comprising a nucleic acid or vector as described herein. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.

[0026] In another aspect, a composition is provided, which comprises one or more of the recombinant collagen, nucleic acid, vector and host cell described herein. In one embodiment, the composition is a kit. In one embodiment, the composition is a biological dressing, a human biomimetic material, a plastic or cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printing artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient and a food additive. In one embodiment, the composition is a surface composition, an injection composition or an oral composition. In one embodiment, the composition is a composition in the form of a solution, a lyophilized powder, a gel, a sponge or a fiber.

[0027] In another aspect, provided herein are uses of the recombinant collagen, nucleic acid, vector, host cell and / or composition in one or more of biological dressings, human bionic materials, plastic or cosmetic 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 printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0028] In another aspect, a method for promoting cell adhesion is provided, comprising contacting a recombinant collagen protein, nucleic acid, vector, host cell, and / or composition herein with a cell. In one embodiment, the cell is an animal cell. The animal cell can be a mammalian cell or a human cell. Provided is the use of the recombinant collagen protein, nucleic acid, vector, host cell, and / or composition herein in preparing a kit for promoting cell adhesion.

[0029] In another aspect, a cosmetic method comprises administering the recombinant collagen described herein to a subject, preferably topically, orally or by injection, preferably the subject is a human.

[0030] In another aspect, a method for producing the recombinant collagen described herein is provided, comprising:

[0031] (1) culturing the host cell described herein under appropriate culture conditions;

[0032] (2) harvesting the host cells and / or culture medium containing the recombinant collagen; and

[0033] (3) Purification of recombinant collagen.

[0034] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21 (DE3) cell.

[0035] In one embodiment, step (1) comprises culturing E. coli cells in LB medium and inducing expression by IPTG.

[0036] In one embodiment, step (2) comprises harvesting the E. coli cells, resuspending them in a balanced working solution, homogenizing the E. coli cells, preferably by high pressure homogenization, and separating the supernatant. In one embodiment, the balanced working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, pH 7-9.

[0037] In one embodiment, step (3) includes crude purification, enzyme digestion, polishing, and / or nickel column purification.

[0038] In one embodiment, the crude purification comprises subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein, wherein the eluate comprises 100-500 mM sodium chloride, 10-50 mM Tris and 100-500 mM imidazole, preferably at pH 7-9.

[0039] In one embodiment, the purification comprises gradient elution of the eluate containing the target protein using a strong anion exchange chromatography column; preferably, the gradient elution comprises 0-15% B solution for 1-5 minutes and then holding for 3 column volumes, 15-30% B solution for 1-5 minutes and then holding for 3 column volumes, 30-50% B solution for 1-5 minutes and then holding for 3 column volumes, and 50-100% B solution for 1-5 minutes and then holding for 3 column volumes; wherein B solution contains 10-50mM Tris, 0.5-5M sodium chloride, pH 7-9. In one embodiment, step (3) comprises purifying the recombinant collagen using a purification column, such as a nickel column, and / or cleaving the recombinant collagen using a collagenase.

[0040] Advantages of the present invention include:

[0041] 1. The recombinant collagen of the present invention is derived from human type IV collagen and is a recombinant type IV humanized collagen;

[0042] 2. The recombinant collagen of the present invention is suitable for the preparation of Escherichia coli and can be separated and purified;

[0043] 3. The recombinant collagen of the present invention has a large yield and is suitable for subsequent purification (Ni column or strong anion column purification); and

[0044] 4. The recombinant collagen of the present invention has cell adhesion activity. The recombinant collagen of the present invention (eg, C4P7Ch) has higher cell adhesion activity than the positive control. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the results of C4P7Ca electrophoresis detection.

[0046] Figure 2 shows the results of electrophoresis detection of C4P7Cb.

[0047] FIG3 shows the electrophoresis detection results of C4P7Cc.

[0048] FIG4 shows the electrophoresis detection results of C4P7Cd.

[0049] FIG5 shows the electrophoresis detection results of C4P7Ce.

[0050] FIG6 shows the electrophoresis detection results of C4P7Cf.

[0051] FIG7 shows the electrophoresis detection results of C4P7Cg.

[0052] FIG8 shows the results of C4P7Ch electrophoresis detection.

[0053] FIG9 shows the electrophoresis detection results of C4P7Ea.

[0054] FIG10 shows the results of electrophoresis detection of C4P7Eb.

[0055] FIG11 shows the electrophoresis detection results of C4P7Ec.

[0056] FIG12 shows the effect of collagen C4P7Ch on cell adhesion.

[0057] FIG13 shows the effect of collagen C4P7Cf on cell adhesion. DETAILED DESCRIPTION

[0058] As used herein, "recombinant collagen" refers to an amino acid sequence or fragment thereof encoded by a specific gene that has been designed and modified and prepared by DNA recombination technology, or a combination of such functional amino acid sequence fragments. The gene coding sequence or amino acid sequence of the recombinant collagen may have low homology to the gene coding sequence or amino acid sequence of human collagen. "Recombinant humanized collagen" refers to a full-length or partial amino acid sequence fragment encoded by a gene of a specific type of human collagen prepared by DNA recombination technology, or a combination of functional fragments of human collagen. In this article, recombinant collagen comprises one or more repeating units. The repeating unit may be derived from human type IV collagen. Therefore, the recombinant collagen may be recombinant type IV humanized collagen. Multiple repeating units may be connected by a linker, which may be the natural amino acid residues of the repeating unit on human type IV collagen, for example, 1-50 amino acid residues. The repeating unit may be SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33. The recombinant collagen may be SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32.

[0059] As used herein, the term "variant" means a recombinant collagen having cell adhesion activity that includes a change / mutation (i.e., substitution, addition, insertion and / or deletion) at one or more positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. Addition refers to the addition of one or more amino acid residues at the C-terminus and / or N-terminus of the amino acid sequence. The substitution may be a conservative substitution. The variant of the repeating unit may be a sequence after one or more amino acid residues are changed or mutated (i.e., substituted, added, inserted and / or deleted) in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33. The variant of the recombinant collagen may be a sequence after one or more amino acid residues are changed or mutated (i.e., substituted, added, inserted and / or deleted) in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32.

[0060] For example, a variant of the repeat unit of SEQ ID NO: 1 may be a variant comprising the following mutations: addition of Gfpgfp (SEQ ID NO: 34) or an N-terminal truncation of SEQ ID NO: 34 to the N-terminus of the amino acid sequence of SEQ ID NO: 1 (i.e., truncation of 1-5 amino acid residues, such as 1, 2, 3, 4 or 5 amino acid residues, corresponding to fpgfp, pgfp, gfp, fp and p residues, respectively, from the N-terminus of SEQ ID NO: 34), and / or addition of GFMGPPGPQGQPGLP (SEQ ID NO: 35) or a C-terminal truncation of SEQ ID NO: 35 to the C-terminus of the amino acid sequence of SEQ ID NO: 1 (truncation of 1-14 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues, from the C-terminus of SEQ ID NO: 35). A variant of the repeat unit of SEQ ID NO: 1 may be a variant comprising the following mutations: addition of Gfpgfp (SEQ ID NO: 34) or an N-terminal truncated fragment thereof (i.e., truncating 1, 2, 3, 4 or 5 from the N-terminus of SEQ ID NO: 34) to the N-terminus of the amino acid sequence of SEQ ID NO: 1, and / or truncating 1-5 amino acid residues, for example 1, 2 or 3 amino acid residues, at the C-terminus of the amino acid sequence of SEQ ID NO: 1.

[0061] A variant of the repeat unit of SEQ ID NO: 28 may be a variant comprising the following mutation: addition of Glpgtp (SEQ ID NO: 36) or an N-terminal truncation thereof to the N-terminus of the amino acid sequence of SEQ ID NO: 28 (i.e., truncation of 1-5 amino acid residues, e.g., 1, 2, 3, 4 or 5 amino acid residues, from the N-terminus of SEQ ID NO: 36).

[0062] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes reflected in one or more of the following tables:

[0063] Conserved amino acid residues:

[0064] Physical and functional classification of alternative amino acid residues:

[0065] As used herein, "cell adhesion" refers to the adhesion between cells and collagen. Collagen (such as the recombinant collagen described herein) can promote the adhesion between cells and the container in which the cells are cultured.

[0066] As used herein, the term "expression" includes any step involved in the production of recombinant collagen including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0067] As used herein, the term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a recombinant collagen and is operably linked to control sequences that provide for its expression.

[0068] As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0069] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified into a nucleic acid-containing segment in a manner not originally found in nature, or is synthetic, and may contain one or more control sequences. The nucleic acid may be SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. The nucleic acid may be a codon-optimized nucleic acid, such as a nucleic acid codon-optimized for expression in E. coli cells.

[0070] The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0071] The degree of association between two amino acid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use as in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, people such as Rice, 2000, Trends Genet. [genetics trend] 16:276-277) (preferred 5.0.0 version or updated version) Ni Deer program implemented Ni Deerman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using non-simplified option to obtain) of Ni Deer, who is labeled as " longest identity ", is used as identity percentage and is calculated as follows:

[0072] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment)

[0073] For purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) as implemented by the Needleman program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000) (preferred 5.0.0 version or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needleman, labeled "longest identity," (obtained using the non-simplified option), which is labeled "longest identity," is used as percent identity and is calculated as follows:

[0074] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0075] Recombinant collagen

[0076] The present invention provides a recombinant collagen comprising one or more repeating units, wherein the repeating units are connected directly or through a linker, and the repeating units comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33. The variant can be (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33, 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% or 100% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33. For the recombinant collagen described herein, the mutation may be selected from substitution, addition, insertion or deletion. Preferably, the substitution is a conservative amino acid substitution.

[0077] The recombinant collagen described herein can comprise a plurality of repeating units, for example, 2-50 repeating units, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 repeating units.

[0078] The linker in the recombinant collagen described herein may comprise one or more amino acid residues, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0079] The recombinant collagen is recombinant human type IV collagen or recombinant type IV humanized collagen, preferably having cell adhesion activity. The recombinant collagen described herein may be derived from human, and thus is recombinant type IV humanized collagen.

[0080] The recombinant collagen described herein may also comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence.

[0081] Nucleic acid constructs

[0082] The present invention also relates to nucleic acid constructs comprising a nucleic acid of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. A vector may comprise a nucleic acid construct.

[0083] Nucleic acids can be manipulated in a variety of ways to provide expression of recombinant collagen. Depending on the expression vector, it may be desirable or necessary to manipulate the nucleic acid prior to its insertion into the vector. Techniques for modifying nucleic acids using recombinant DNA methods are well known in the art.

[0084] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of the recombinant collagen protein of the present invention. The promoter comprises transcriptional control sequences that mediate the expression of the recombinant collagen protein. The promoter can be any nucleic acid that exhibits transcriptional activity in the host cell, including variants, truncated forms, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular recombinant collagen protein that is homologous or heterologous to the host cell.

[0085] Examples of suitable promoters for directing transcription of the vectors or nucleic acid constructs of the present invention in bacterial host cells are those obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltoamylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene, the Escherichia coli lac operon, the Escherichia coli trc promoter.

[0086] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

[0087] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the recombinant collagen. Any terminator that is functional in the host cell can be used in the present invention.

[0088] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0089] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. (1992).

[0090] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0091] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0092] The control sequence may also be a leader sequence, which is a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the recombinant collagen. Any leader sequence that is functional in the host cell may be used.

[0093] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0094] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0095] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0096] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of the recombinant collagen and instructs the recombinant collagen to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the recombinant collagen in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the recombinant collagen. However, any signal peptide coding sequence that instructs the expressed recombinant collagen to enter the secretory pathway of the host cell can be used.

[0097] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0098] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (Yeast 8:423-488).

[0099] expression vector

[0100] The present invention further relates to a recombinant expression vector comprising nucleic acid of the present invention, promoter and transcription and translation termination signals. Nucleic acid and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to insert or replace the polynucleotide encoding the recombinant collagen at such sites. Alternatively, the polynucleotide may be expressed by inserting a nucleic acid or a nucleic acid construct comprising the nucleic acid into a suitable vector for expression. When producing an expression vector, the coding sequence is positioned in the vector so that the coding sequence is operably connected to the suitable control sequence for expression.

[0101] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0102] The carrier can be an autonomous replicating vector, i.e. a carrier existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can contain any means for ensuring self-replication. Alternatively, the carrier can be a carrier that is integrated into the genome and replicates with one or more chromosomes into which it has been integrated when it is introduced into the host cell. Moreover, a single carrier or plasmid or two or more carriers or plasmids can be used that contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0103] The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0104] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0105] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is the hph-tk dual selectable marker system.

[0106] The vector may contain elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0107] For being integrated into the host cell genome, vector can rely on the polynucleotide sequence encoding this recombinant collagen or any other element of this vector for being integrated into this genome by homologous or non-homologous recombination.Alternatively, vector can contain other polynucleotides at the precise position in the chromosome that is integrated into the host cell genome by homologous recombination for guiding.In order to improve the possibility of integration at the precise position, integration element should contain a sufficient number of nucleic acids, for example 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, and these nucleic acids have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination.Integration element can be any sequence homologous to the target sequence in the host cell genome.And, integration element can be non-coding or encoded polynucleotide.On the other hand, vector can be integrated into the genome of the host cell by non-homologous recombination.

[0108] For autonomous replication, the vector may further comprise an origin of replication that enables autonomous replication of the vector in the host cell in question. The origin of replication may be any plasmid replicator that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0109] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0110] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0111] More than one copy of a polynucleotide of the present invention can be inserted into a host cell to increase the production of recombinant collagen. Increased copy numbers of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0112] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).

[0113] host cells

[0114] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the recombinant collagen of the present invention. A construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend largely on the gene encoding the recombinant collagen and its source.

[0115] The host cell can be any cell useful in the recombinant production of the recombinant collagen of the present invention, for example, a prokaryotic or eukaryotic cell.

[0116] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0117] Host cells can also be eukaryotic, such as mammalian, insect, plant or fungal cells. Plant cells herein do not include plant cells that can be regenerated into plants. Animal cells also do not include cells that can produce animal bodies.

[0118] The host cell can be a fungal cell, such as Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota, etc. The fungal host cell can be a yeast cell, including ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0119] Production method

[0120] The present invention also relates to a method for producing the recombinant collagen described herein, comprising:

[0121] (1) culturing the host cell described herein under appropriate culture conditions;

[0122] (2) harvesting the host cells and / or culture medium containing the recombinant collagen; and

[0123] (3) Purification of recombinant collagen.

[0124] Host cell is cultivated in the nutrient medium of suitable method known in the art production recombinant collagen.For example, can be by shake flask culture or in laboratory or industrial fermenter small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) culture cell, this culture is in suitable medium and under the condition that recombinant collagen is expressed and / or separated, is carried out.Using program as known in the art, cultivation occurs in the suitable nutrient medium comprising carbon and nitrogen source and inorganic salt.Suitable culture medium can be obtained from commercial supplier or can be prepared according to disclosed composition (for example, in the catalogue of American Type Culture Collection).If recombinant collagen is secreted into this nutrient medium, then this recombinant collagen can be directly recovered from this culture medium.If recombinant collagen is not secreted, then it can be recovered from cell lysate.

[0125] Recombinant collagen can be detected using methods known in the art that are specific for recombinant collagen. These detection methods include, but are not limited to, the use of specific antibodies. For example, adhesion assays can be used to determine the activity of recombinant collagen.

[0126] Recombinant collagen can be recovered using methods known in the art. For example, recombinant collagen can be recovered from the nutrient medium by conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the fermentation broth containing the recombinant collagen is recovered.

[0127] Recombinant collagen can be purified by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, in order to obtain substantially pure recombinant collagen.

[0128] Step (1) may include one or more of the following steps: constructing an expression plasmid, for example, inserting the coding nucleotide sequence into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid. The successfully constructed expression plasmid can be transformed into Escherichia coli cells (for example, Escherichia coli competent cells BL21 (DE3)). The specific process can be: (1) taking the plasmid to be transformed and adding it to the Escherichia coli competent cells BL21 (DE3); (2) placing the mixture in an ice bath (for example, 10-60 minutes, for example, 30 minutes), then heat shocking it in a water bath (for example, at 40-50°C, for example, 42°C, for 45-90 seconds), taking it out and placing it in an ice bath (for example, 1-5 minutes, for example, 2 minutes); (3) adding liquid LB culture medium, and then culturing (for example, at 35-40°C, for example, 37°C, 150-300 rpm, for example, 220 rpm, for 40-80 minutes, for example, 60 minutes); (4) applying the bacterial solution and selecting single colonies. For example, take the bacterial solution and spread it evenly on an LB plate containing ampicillin sodium, and culture the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0129] Step (2) may include culturing a single colony in LB medium containing an antibiotic stock solution (e.g., at 150-300 rpm, e.g., 220 rpm, 35-40° C., e.g., 37° C., in a constant temperature shaker for 5-10 h, e.g., 7 h). The cultured shake flask is then cooled to 10-20° C., e.g., 16° C., and IPTG is added to induce expression for a period of time before collecting the cells (e.g., by centrifugation).

[0130] Step (3) may include resuspending the cells in a balanced working solution, cooling the solution to ≤15°C, homogenizing (e.g., high pressure homogenization, e.g., 1-5 times, e.g., 2 times), and separating the homogenized solution to obtain a supernatant. The balanced working solution may comprise 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, with a pH of 7-9. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0131] Step (3) may include purifying and enzymatically cleaving the recombinant collagen. The purification may be crude purification, including subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein. Crude purification may include washing the column material with water, for example, 2-10 column volumes (CV), for example, 5 CV. The column material may be balanced with an equilibrium solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), for example, 2-10 CV, for example, 5 CV. The equilibrium solution may comprise 100-500 mM sodium chloride, 10-50 mM Tris and 10-50 mM imidazole, pH 7-9. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0132] Step (3) may include adding the supernatant to the column material and washing the impurity protein with a washing solution. The washing solution may comprise 100-500 mM sodium chloride, 10-50 mM Tris and 10-50 mM imidazole, pH 7-9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH can be 7, 7.5, 8, 8.5, or 9. Then, an eluent can be added and the flow-through can be collected. The eluent can comprise 100-500 mM sodium chloride, 10-50 mM Tris, 100-500 mM imidazole, pH 8.0. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The pH can be 7, 7.5, 8, 8.5, or 9.

[0133] Enzymatic digestion can include adding collagenase for digestion (based on a ratio of total protein to total collagenase of 10-100:1, e.g., 50:1, and digestion at 10-20°C, e.g., 16°C, for 2-8 hours, e.g., 4 hours). The protein solution after digestion is dialyzed, e.g., placed in a dialysis bag, dialyzed at 1-6°C, e.g., 4°C, for 1-8 hours, e.g., 2 hours, and then transferred to new dialysate for overnight dialyzation at 1-6°C, e.g., 4°C.

[0134] Purification may include polishing (e.g., protein isoelectric point > 8.0). Preferably, polishing comprises gradient elution of the eluate containing the target protein or the product after enzymatic digestion (e.g., the product after enzymatic digestion and dialysis) using a strong anion exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). The gradient elution comprises 0-15% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes, 15-30% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes, 30-50% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes, 50-100% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes. Solution B may comprise 10-50 mM Tris, 0.5-5 M sodium chloride, pH 7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM. The concentration of sodium chloride is 1, 2, 3, or 4 M. The pH can be 7, 7.5, 8, 8.5, or 9. Purification can include equilibrating the column with Solution A and loading the sample, followed by gradient elution. Solution A can comprise 10-50 mM Tris, 10-50 mM sodium chloride, and a pH of 7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM. The concentration of sodium chloride is 15, 20, 25, 30, 35, 40, or 45 mM. The pH can be 7, 7.5, 8, 8.5, or 9.

[0135] 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.

[0136] The following examples are provided to further illustrate the present invention.

[0137] Example

[0138] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In conjunction with this specification and common knowledge in the art, a person of ordinary skill in the art will clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0139] Example 1: Construction, expression and screening of recombinant humanized type IV collagen fragments

[0140] 1. Perform large-scale functional region screening to obtain the following different target gene functional regions of recombinant humanized type IV collagen.

[0141] 1) Amino acid sequence of C4P7Ch (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeq, SEQ ID NO: 1, the number of repeating units is 10; the amino acid sequence of C4P7Ch is SEQ ID NO: 2):

[0142] C4P7Ch base sequence (SEQ ID NO: 3):

[0143] 2) C4P7Cf (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, SEQ ID NO: 4, the number of repeating units is 8; the amino acid sequence of C4P7Cf is SEQ ID NO: 5):

[0144] C4P7Cf base sequence (SEQ ID NO: 6):

[0145] 3) C4P7Ca amino acid sequence (the amino acid sequence of the repeating unit is GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP, SEQ ID NO: 7, the number of repeating units is 6; the amino acid sequence of C4P7Ca is SEQ ID NO: 8):

[0146] C4P7Ca base sequence (SEQ ID NO: 9):

[0147] 4) C4P7Cb amino acid sequence (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, SEQ ID NO: 10, the number of repeating units is 8; the amino acid sequence of C4P7Cb is SEQ ID NO: 11):

[0148] C4P7Cb base sequence (SEQ ID NO: 12):

[0149] 5) C4P7Cc amino acid sequence (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm, SEQ ID NO: 13, the number of repeating units is 8; the amino acid sequence of C4P7Cc is SEQ ID NO: 14):

[0150] C4P7Cc base sequence (SEQ ID NO: 15):

[0151] 6) C4P7Cd amino acid sequence (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeq, SEQ ID NO: 16, the number of repeating units is 10; the amino acid sequence of C4P7Cd is SEQ ID NO: 17):

[0152] C4P7Cd base sequence (SEQ ID NO: 18):

[0153] 7) C4P7Ce amino acid sequence (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp, SEQ ID NO: 19, the number of repeating units is 8; the amino acid sequence of C4P7Ce is SEQ ID NO: 20):

[0154] C4P7Ce base sequence (SEQ ID NO: 21):

[0155] 8) C4P7Cg amino acid sequence (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfm, SEQ ID NO: 22, the number of repeating units is 10; the amino acid sequence of C4P7Cg is SEQ ID NO: 23):

[0156] C4P7Cg base sequence (SEQ ID NO: 24):

[0157] 9) C4P7Ea amino acid sequence (the amino acid sequence of the repeating unit is Glpgtpgptgpagqkgepgsdgipgsagekgepglp, SEQ ID NO: 25, the number of repeating units is 10; the amino acid sequence of C4P7Ea is SEQ ID NO: 26):

[0158] C4P7Ea base sequence (SEQ ID NO: 27):

[0159] 10) C4P7Eb amino acid sequence (the amino acid sequence of the repeating unit is Gptgpagqkgepgsdgipgsagekgepglp, SEQ ID NO: 28, the number of repeating units is 10; the amino acid sequence of C4P7Eb is SEQ ID NO: 29):

[0160] C4P7Eb base sequence (SEQ ID NO: 30):

[0161] 11) C4P7Ec amino acid sequence (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgsk, SEQ ID NO: 31, the number of repeating units is 10; the amino acid sequence of C4P7Ec is SEQ ID NO: 32):

[0162] C4P7Ec base sequence (SEQ ID NO: 33):

[0163] Each of the above encoding nucleotide sequences was commercially synthesized. Each of the above encoding nucleotide sequences (with a collagenase cleavage site added at the 5' end, the amino acid sequence of the collagenase cleavage site being ENLYFQ and the nucleotide sequence being GAAAACCTGTATTTCCAG) was inserted between the KpnI and XhoI restriction sites of the pET-28a-Trx-His expression vector to generate a recombinant expression plasmid.

[0164] 3. Transform the successfully constructed expression plasmid 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 ampicillin sodium. (5) Incubate the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0165] 4. Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing LB medium containing antibiotic stock solution. Incubate in a shaker at 220 rpm and 37°C for 7 hours. Cool the shake flask to 16°C and add IPTG to induce expression for a period of time. Then, divide the culture into centrifuge bottles and centrifuge at 8000 rpm and 4°C for 10 minutes. Collect the cells and record the cell weight. Take a sample (labeled "cell solution") for electrophoresis.

[0166] 5. Resuspend the collected cells in a balanced working solution (200mM NaCl, 25mM Tris, 20mM imidazole, pH 8.0). Cool the solution to ≤15°C and homogenize. High-pressure homogenize twice (take samples from each homogenization and label them as "homogeneous first" and "homogeneous second"). After completion, collect the solution. Aliquot the homogenized solution into centrifuge bottles and centrifuge at 17,000 rpm and 4°C for 30 minutes. Collect the supernatant and perform electrophoresis on the supernatant (labeled "supernatant") and the precipitate.

[0167] 6. Recombinant humanized type IV collagen was purified and enzymatically digested. The specific process was as follows: (1) Crude purification: a. Wash the column (Ni6FF, Cytiva) with water for 5 CV. b. Equilibrate the column with equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CV. c. Loading: Add the supernatant after centrifugation to the column until the liquid has flowed out, and then take the flow-through for electrophoresis (marked as flow-through). d. Cleaning impurities: Add 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid has flowed out, and then take the wash flow-through for electrophoresis (marked as wash impurities). e. Collect the target protein: add 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0), and collect the flow-through (marked as: elution), detect the protein concentration, calculate the protein amount, and perform electrophoresis detection. f. Wash the column with 1 M imidazole working solution (marked as 1 M wash). g. Wash the column with purified water. (2) Enzyme digestion: add TEV enzyme at a ratio of 50:1 between the total amount of protein and the total amount of TEV enzyme, digest at 16°C for 4 hours, and take a sample for electrophoresis detection (marked as after digestion). Place the protein solution after enzyme digestion in a dialysis bag, dialyze at 4°C for 2 hours, and then transfer to new dialysate for overnight dialyzation at 4°C (marked as liquid change).

[0168] (3) Purification: a. Equilibrate the column (Capto Q, Cytiva): Use solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) to equilibrate the column at a flow rate of 10 ml / min. b. Load the sample: The flow rate is 5 ml / min, load the sample and collect the flow-through (marked as QFL), and perform electrophoresis detection. c. Gradient elution: Set 0-15% solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 min and then maintain for 3 CVs, 15-30% solution B for 2 min and then maintain for 3 CVs, 30-50% solution B for 2 min and then maintain for 3 CVs, 50-100% solution B for 2 min and then maintain for 3 CVs, collect the peaks and perform electrophoresis detection (marked as B wash). d. Wash the column. Store the protein at 4°C.

[0169] 7. Electrophoresis detection

[0170] The specific process is as follows: take 40 μl of sample liquid, add 10 μl of 5× protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in boiling water at 100°C for 10 minutes, then add 10 μl to each well of SDS-PAGE protein gel, run at 80 V for 2 hours, and then use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20 minutes, and then use protein decolorization solution (10% acetic acid, 5% ethanol) for decolorization.

[0171] Figure 1 shows the electrophoresis detection results of C4P7Ca. Figure 2 shows the electrophoresis detection results of C4P7Cb. Figure 3 shows the electrophoresis detection results of C4P7Cc. Figure 4 shows the electrophoresis detection results of C4P7Cd. Figure 5 shows the electrophoresis detection results of C4P7Ce. Figure 6 shows the electrophoresis detection results of C4P7Cf. Figure 7 shows the electrophoresis detection results of C4P7Cg. Figure 8 shows the electrophoresis detection results of C4P7Ch. Figure 9 shows the electrophoresis detection results of C4P7Ea. Figure 10 shows the electrophoresis detection results of C4P7Eb. Figure 11 shows the electrophoresis detection results of C4P7Ec. Figures 1 to 11 show that the actual molecular weights of the isolated proteins (C4P7Ca, C4P7Cb, C4P7Cc, C4P7Cd, C4P7Ce, C4P7Cf, C4P7Cg, C4P7Ch, C4P7Ea, C4P7Eb and C4P7Ec) are consistent with their corresponding expected molecular weights, indicating that the proteins are correctly expressed.

[0172] Example 2: Mass spectrometry detection of recombinant humanized type IV collagen

[0173] Experimental methods

[0174] Protein samples (C4P7Cf and C4P7Ch collagen) were reduced with DTT and alkylated with iodoacetamide before being digested with trypsin overnight. The resulting peptides were desalted using a C18ZipTip and then mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) and spotted on a plate. Finally, the peptides were analyzed using a matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF / TOF) using an Ulraflextreme. TM , Brucker, Germany for analysis (the technology of peptide fingerprinting can be found in Protein J. 2016; 35: 212-7).

[0175] Data retrieval was performed using the MS / MS Ion Search page on the local Masco website. Protein identification was based on the primary mass spectra of peptides generated after enzymatic digestion. Detection parameters: Trypsin digestion with two missed cleavage sites. Alkylation of cysteine ​​was set as a fixed modification. Oxidation of methionine was set as a variable modification. The NCBprot database was used for identification.

[0176] Table 1: Mass spectrometry-detected molecular weights and corresponding peptides of recombinant humanized type IV collagen C4P7Cf

[0177] The coverage of the detected peptide fragments compared with the theoretical sequence was 99.04% The underlined residues are covered parts), and the detection results are very reliable.

[0178] Table 2: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant humanized type IV collagen C4P7Ch

[0179] The coverage of the detected peptide fragments compared with the theoretical sequence was 98% The underlined residues are covered parts), and the detection results are very reliable.

[0180] Example 3: Bioactivity Detection of Recombinant Humanized Type IV Collagen

[0181] 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:

[0182] (1) The concentration of the protein sample 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 humanized collagen C4P7Cf and C4P7Ch provided by the present invention.

[0183] Specifically, the sample's UV absorbance at 215 nm and 225 nm is measured, and the protein concentration is calculated using the empirical formula C (μg / mL) = 144 × (A215 - A225). Note that the assay must be performed when A215 < 1.5. This method, which measures the characteristic absorption of peptide bonds under far-UV light, is unaffected by chromophore content, exhibits minimal interfering substances, and is simple to perform. It is suitable for detecting human collagen and its analogs, which do not develop color with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.) After measuring the protein concentration, adjust the concentration of all tested proteins to 0.5 mg / mL with PBS.

[0184] (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).

[0185] (3) Coating: Different concentrations of collagen (C4P7Cf or C4P7Ch) and positive and negative controls were added to the ELISA plate, 100 μL per well, 5 replicate wells per group, and incubated at 4°C overnight.

[0186] (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.

[0187] (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.

[0188] (6) Detection: Use CCK8 detection kit (manufacturer: Beyotime, product catalog number C0038) to detect OD 450 The absorbance is measured in nm. The degree of cell adhesion is calculated using the following formula. The cell adhesion rate reflects the cell adhesion capacity of collagen. The higher the cell adhesion capacity, the more quickly the cells are provided with a high-quality external environment, helping them adhere.

[0189] Where:

[0190] P: relative cell adhesion ratio;

[0191] OD1: The average value of UV absorbance of each replicate well of the collagen sample at 450 nm;

[0192] OD2: average UV absorbance of each replicate well of the control collagen sample at 450 nm;

[0193] OD0: The average value of UV absorbance of each replicate well in the blank control group at 450 nm.

[0194] (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.

[0195] The results are shown in Figures 11 and 12. Compared with the D-PBS group, the positive control group had a significant effect on promoting cell adhesion. Recombinant humanized collagen C4P7Cf and C4P7Ch also promoted cell adhesion.

[0196] Although the present invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions, and / or additions may be made and that substantial equivalents may be substituted for elements of the described embodiments without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from the scope of the invention. Therefore, it is not intended that the present invention be limited to the specific embodiments disclosed for carrying out the invention, but rather that the invention will encompass all embodiments falling within the scope of the appended claims.

Claims

1. A recombinant collagen comprising one or more repeating units, wherein the repeating units are connected directly or through a linker, wherein the repeating units comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1 or 28, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence 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% or 100% identity with the amino acid sequence; Preferably, the plurality of repeating units is 2-50 repeating units, such as 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 4-10 or 6-10 repeating units; Preferably, wherein the linker comprises one or more amino acid residues, such as 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acid residues; Preferably, wherein the mutation is selected from substitution, addition, insertion or deletion; Preferably, wherein the substitution is a conservative amino acid substitution; Preferably, the recombinant collagen is recombinant humanized collagen; preferably recombinant type IV collagen; preferably recombinant human type IV collagen or recombinant type IV humanized collagen; Preferably, the recombinant collagen has cell adhesion activity; Preferably, the variant of SEQ ID NO: 1 comprises the following mutations: adding GFPGFP (SEQ ID NO: 34) or an N-terminal truncated fragment of 1-5 amino acid residues of SEQ ID NO: 34 to the N-terminus of the amino acid sequence of SEQ ID NO: 1, and / or adding GFMGPPGPQGQPGLP (SEQ ID NO: 35) or a C-terminal truncated fragment of 1-14 amino acid residues of SEQ ID NO: 35 to the C-terminus of the amino acid sequence of SEQ ID NO: 1, or truncating 1-5 amino acid residues at the C-terminus of the amino acid sequence of SEQ ID NO: 1; Preferably, the variant of SEQ ID NO: 28 comprises the following mutations: adding Glpgtp (SEQ ID NO: 36) or an N-terminal truncated fragment thereof of 1 to 5 amino acid residues in length to the N-terminus of the amino acid sequence of SEQ ID NO: 28; Preferably, the variant of SEQ ID NO: 1 is selected from SEQ ID NO: 4, 7, 10, 13, 16, 19, 22, or 31; and / or the variant of SEQ ID NO: 28 is SEQ ID NO:

25.

2. The recombinant collagen according to claim 1, comprising an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence 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% or 100% identity with the amino acid sequence; Preferably, wherein the mutation is selected from substitution, addition, insertion or deletion; Preferably, the substitution is a conservative amino acid substitution.

3. A nucleic acid encoding the recombinant collagen according to claim 1 or 2, Preferably, wherein the nucleic acid comprises a codon-optimized nucleotide sequence, Preferably, the nucleotide sequence is a nucleotide sequence codon-optimized for expression in eukaryotic host cells or prokaryotic host cells, such as yeast or Escherichia coli; Preferably, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33.

4. A vector comprising the nucleic acid according to claim 3, Preferably, the vector comprises an expression control element, a purification tag nucleotide and / or a leader sequence nucleotide operably linked to the nucleic acid, Preferably, wherein the expression control element is selected from a promoter, a terminator or an enhancer; Preferably, the purification tag is selected from a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag; Preferably, the vector is an expression vector or a cloning vector, preferably pET-28a(+).

5. A host cell comprising a nucleic acid according to claim 3 or a vector according to claim 4; wherein preferably, the host cell is a eukaryotic cell or a prokaryotic cell; wherein preferably, the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.

6. A composition comprising one or more of the recombinant collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4 and the host cell according to claim 5, preferably, the composition is a kit; preferably, the composition is one or more of biological dressings, human bionic materials, plastic or cosmetic 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 solution, a lyophilized powder, a gel, a sponge or a fiber.

7. Use of the recombinant collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5 and / or the composition according to claim 6 in one or more of biological dressings, human bionic materials, plastic or cosmetic 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.

8. A method for promoting cell adhesion, comprising the step of contacting a cell with the recombinant collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5 and / or the composition according to claim 6, wherein the cell is an animal cell, preferably a mammalian cell, preferably a human cell.

9. A cosmetic method, comprising administering the recombinant collagen according to claim 1 or 2 to a subject, preferably the administration is topical administration, oral administration or injection administration; preferably, the subject is a human.

10. A method for producing the recombinant collagen according to claim 1 or 2, comprising: (1) culturing the host cell according to claim 5 under suitable culture conditions; (2) harvesting host cells and / or culture medium containing the recombinant collagen; and (3) purifying recombinant collagen; Preferably, the host cell is an Escherichia coli cell, preferably an Escherichia coli BL21 (DE3) cell; Preferably, step (3) comprises purifying the recombinant collagen using a purification column, such as a nickel column, and / or cleaving the recombinant collagen using a collagenase.

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