Recombinant collagen, nucleic acid, vector, host cell, composition, use of recombinant collagen, nucleic acid, vector, host cell and / or composition, and, in vitro method for facilitating cell adhesion for non-therapeutic purposes, and for producing collagen recombinant

Description

55 Recombinant collagen, nucleic acid, vector, host cell, composition, use of recombinant collagen, nucleic acid, vector, host cell and / or composition, and in vitro method to facilitate cell adhesion for non-therapeutic purposes, and to produce recombinant collagen.

[001] This application claims priority over Chinese Invention Patent Application No. 202311391711.1, filed on October 25, 2023, entitled “A Method for Preparing Biosynthetic Human Structural Material Type IV Collagen”. Technical Field

[002] The present description refers to the field of biomedicine and refers to a recombinant humanized type IV collagen, as well as its method of preparation and use. Fundamentals of Description

[003] Collagen (abbreviated as COL) is a functional fibrous helical protein consisting of three peptide chains. As a major component of the extracellular matrix, it is abundant in quantity and widely distributed. In the human body, collagen represents 25% to 30% of the total protein content and is mainly present in the skin, tendons, and bones. It plays a crucial role in the protection and connection of various tissues, performing significant physiological functions in vivo. Type IV collagen is an essential protein that, together with laminin, constitutes the basement membrane. Its structural unit is a trimer with a helical structure composed of three α chains. Although the trimer subunits vary depending on the tissue, in several tissues, such as the liver, the trimer consists of two α1 chains and one α2 chain. The type IV collagen molecule has unique structures at both ends of the TH domain that form the helical structure.The N-terminal structure is designated as... Petition 870260016232, dated 20 / 02 / 2026, page 17 / 126 / 55 domain 7S, and the C-terminal structure is called domain NC1.

[004] In recent years, collagen has gained significant attention as a research focus due to its favorable biocompatibility, degradability, low antigenicity, and unique biological structure. It finds wide applications in multiple fields, such as biomedicine, cosmetics, health products, and food.

[005] There are 28 different types of collagen found in the human body, which can be classified into two main categories: fibrous collagen and non-fibrous collagen, depending on whether their structure is fibrous or not. Fibrous collagen primarily performs the function of a cellular scaffold, fixing cell positions and acting as anchors, while also providing tensile strength and rigidity to tissues. Non-fibrous collagen, on the other hand, is further subdivided into basal collagen, short-chain collagen, transmembrane collagen, etc., each performing distinct functions.

[006] The traditional method for producing collagen involves purifying animal-derived tissues through acid hydrolysis, alkaline hydrolysis, or enzymatic hydrolysis to extract collagen derivatives. However, collagen obtained by these methods often loses its original biological activity, exhibits low water solubility, has difficulty binding to the human body, faces challenges in avoiding the risks of viral infection and sensitization, and is unable to fully perform its true function. Some research institutions have attempted to express human collagen in vitro using conventional recombinant expression methods, but these approaches are expensive, time-consuming, and unsuitable for large-scale production.

[007] Therefore, there is an urgent market demand for a collagen material with excellent biomaterial properties, a high degree of amino acid sequence homology with the human body, and the ability Petition 870260016232, dated 20 / 02 / 2026, page 18 / 126 / 55 regarding large-scale preparation within an industrial system. Summary of the Invention

[008] The inventors performed a large-scale screening of the functional region of human type IV collagen and identified 11 recombinant collagens. These recombinant collagens can be expressed in Escherichia coli and subsequently purified. Furthermore, the inventors found that these recombinant collagens exhibit higher yields, greater purity of the collagens after fine purification, and greater activity compared to the positive control (bovine type I collagen) in cell adhesion activity assays.

[009] In one aspect, the present description provides a recombinant collagen comprising one or more repeat units linked directly or via a linker, wherein the repeat unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (Gakgdkgskgevgfpglagspgipgskgeq) or 28 (Gptgpagqkgepgsdgipgsagekgepglp), or a variant thereof, and wherein the variant is (1) an amino acid sequence having one or more amino acid residue mutations in said 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 so-called sequence. of amino acids.

[0010] In one modality, the number of repeating units is from 2 to 50 repeating units, such as 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 4 to 10, or 6 to 10 repeating units. For example, the number of repeating units could be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50, or vary between them.

[0011] In one embodiment, the linker comprises one or more amino acid residues, such as 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues. Petition 870260016232, dated 20 / 02 / 2026, p. 19 / 126 / 55

[0012] In one embodiment, the mutation is selected from the group that consists of replacement, addition, insertion, or deletion.

[0013] In one embodiment, the substitution is a conservative amino acid substitution.

[0014] In one embodiment, recombinant collagen is recombinant human type IV collagen or recombinant humanized type IV collagen.

[0015] In one embodiment, recombinant collagen has cell adhesion activity.

[0016] In one embodiment, the variant of SEQ ID NO: 1 comprises the following mutations: the addition of Gfpgfp (SEQ ID NO: 34) or the N-terminal truncated fragment of SEQ ID NO: 34 with a length of 1 to 5 amino acid residues to the N-terminus of the amino acid sequence of SEQ ID NO: 1, and / or the addition of GFMGPPGPQGQPGLP (SEQ ID NO: 35) or the C-terminal truncated fragment of SEQ ID NO: 35 with a length of 1 to 14 amino acid residues to the C-terminus of the amino acid sequence of SEQ ID NO: 1, or the truncation of the amino acid sequence of SEQ ID NO: 1 at the C-terminus by 1 to 5 amino acid residues.

[0017] In one embodiment, the variant of SEQ ID NO: 28 comprises the following mutations: the addition of Glpgtp (SEQ ID NO: 36) or the N-terminal truncated fragment thereof with a length of 1 to 5 amino acid residues to the N-terminal of the amino acid sequence of SEQ ID NO: 28.

[0018] In one embodiment, the variant with SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 4 (Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), 7 (GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQ PGLP), 10 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), 13 Petition 870260016232, dated 20 / 02 / 2026, p. 20 / 126 / 55 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm),16 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeq),19 (Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp),22 (Gakgdkgskgevgfpglagspgipgskgeqgfm) ou31 (Gfpgfpgakgdkgskgevgfpglagspgipgsk).

[0019] In one embodiment, the variant of SEQ ID NO: 28 is SEQ ID NO: 25 (Glpgtpgptgpagqkgepgsdgipgsagekgepglp).

[0020] In one embodiment, recombinant collagen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 and 32, or a variant thereof, wherein the variant is (1) an amino acid sequence having one or more amino acid residue mutations in said 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 said amino acid sequence.

[0021] In one embodiment, the mutation is selected from the group consisting of a substitution, an addition, an insertion, or a deletion. In one embodiment, the substitution is a conservative amino acid substitution.

[0022] In another aspect, a nucleic acid encoding recombinant collagen described in this document is provided. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, the nucleotide sequence is a codon-optimized nucleotide sequence for expression in eukaryotic host cells or prokaryotic host cells, 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.

[0023] In another aspect, a vector is provided that comprises a Petition 870260016232, dated 20 / 02 / 2026, p. 21 / 126 / 55 nucleic acid described in this document. In one embodiment, the vector comprises an expression control element, nucleotides of a purification tag and / or nucleotides of a leader sequence operatively linked to the nucleic acid. In one embodiment, the expression control element is selected from the group consisting of a promoter, a terminator or an enhancer. In one embodiment, the purification tag is selected from the group consisting of 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 a HIS, thrombin or T7 protein tag at the N-terminus and a His tag at the C-terminus. In the present application, recombinant collagen may comprise an enzyme cleavage site at the N-terminal to facilitate purification.

[0024] In another aspect, a host cell comprising a nucleic acid or a vector described in this document is provided. 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 and, in one embodiment, the prokaryotic cell is an Escherichia coli cell, such as E. coli BL21.

[0025] In another aspect, a composition is provided comprising one or more of the recombinant collagens, nucleic acid, vector and host cell described in this document. In one embodiment, the composition is a kit. In one embodiment, the composition is one or more of a biological dressing, a human bionic material, a plastic surgery material or beautification material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filler material, an ophthalmic material, a biomaterial for obstetrics and gynecology, a repair and regeneration material of Petition 870260016232, dated 20 / 02 / 2026, page 22 / 126 / 55 nerves, a material for liver tissue and material for repair and regeneration of blood vessels, a biomaterial for a 3D-printed artificial organ, a cosmetic raw material, a pharmaceutical excipient and a food additive. In one embodiment, the composition is a composition for topical use, injectable use or oral use. In one embodiment, the composition is a composition in the form of a solution, a lyophilized powder, a gel, a sponge or a fiber.

[0026] In another aspect, the use of recombinant collagen, nucleic acid, vector, host cell and / or the composition of the present application is provided for in one or more of the following: a biological dressing, a human bionic material, a material for plastic or aesthetic surgery, an organoid culture material, a cardiovascular stent material, a coating material, a filler material for tissue injection, an ophthalmic material, a biomaterial for obstetrics and gynecology, a material for nerve repair and regeneration, a material for liver tissue and material for blood vessel repair and regeneration, a biomaterial for a 3D printed artificial organ, a cosmetic raw material, a pharmaceutical excipient and a food additive.

[0027] In another aspect, a method is provided for promoting cell adhesion, comprising a step of bringing recombinant collagen, nucleic acid, vector, host cell and / or the composition of the present application into contact with a cell. In one embodiment, the cell is an animal cell. The animal cell may be a mammalian cell or a human cell. The use of recombinant collagen, nucleic acid, vector, host cell and / or the composition of the present application in the manufacture of a kit to facilitate cell adhesion is provided.

[0028] In another aspect, a beautification method is provided which comprises the administration of recombinant collagen described in this Petition 870260016232, dated 20 / 02 / 2026, page 23 / 126 / 55 document to an individual, in which, preferably, the administration is topical, oral or injection administration, preferably, the individual is a human being.

[0029] In another aspect, a method is provided for producing the recombinant collagen described in this document, comprising: (1) incubate the host cell described in this document under suitable culture conditions; (2) collect the host cell and / or culture medium comprising recombinant collagen; and (3) purify the recombinant collagen.

[0030] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21 (DE3) cell.

[0031] In one embodiment, step (1) comprises culturing E. coli cells in LB medium and inducing expression with IPTG.

[0032] In one embodiment, step (2) comprises collecting E. coli cells, resuspending them in an equilibrium working solution, homogenizing (preferably high-pressure homogenization) the E. coli cells and isolating the supernatant. In one embodiment, the equilibrium working solution comprises 100 to 500 mM sodium chloride, 10 to 50 mM Tris, 10 to 50 mM imidazole, at pH 7 to 9.

[0033] In one embodiment, step (3) comprises crude purification, enzymatic digestion, fine purification and / or reverse nickel column purification.

[0034] In one embodiment, crude purification comprises purification on a Ni-agarose resin column of the supernatant to obtain an eluate containing the target protein, wherein the eluate contains 100 to 500 mM sodium chloride, 10 to 50 mM Tris and 100 to 500 mM imidazole, preferably at pH 7 to 9. Petition 870260016232, dated 20 / 02 / 2026, page 24 / 126 / 55

[0035] In one embodiment, fine purification comprises gradient elution of the eluate containing the target protein with a strong anion-exchange chromatography column; wherein, preferably, the gradient elution comprises 0 to 15% solution B for 1 to 5 minutes, followed by retention for 3 column volumes, 15 to 30% solution B for 1 to 5 minutes, followed by retention for 3 column volumes, 30 to 50% solution B for 1 to 5 minutes, followed by retention for 3 column volumes, 50 to 100% solution B for 1 to 5 minutes, followed by retention for 3 column volumes; wherein solution B contains 10 to 50 mM Tris, 0.5 to 5 M sodium chloride, at pH 7 to 9. In one embodiment, step (3) comprises purifying recombinant collagen with a purification column, such as a nickel column, and / or cleaving recombinant collagen with a collagen processing enzyme.

[0036] The advantages of this description include: 1. The recombinant collagen described herein is derived from human type IV collagen and is recombinant humanized type IV collagen; 2. The recombinant collagen described herein is suitable for production by E. coli and can be isolated and purified; 3. The recombinant collagen described herein is produced in high yields and is suitable for subsequent purification (purification on a Ni column or strong anion column); and 4. The recombinant collagen described here has cell adhesion activity. The recombinant collagen described here (e.g., C4P7Ch) has greater cell adhesion activity compared to the positive control. Brief Description of the Drawings

[0037] Figure 1 shows the results of C4P7Ca electrophoresis detection.

[0038] Figure 2 shows the results of detection by electrophoresis. Petition 870260016232, dated 20 / 02 / 2026, page 25 / 126 / 55 of C4P7Cb.

[0039] Figure 3 shows the results of C4P7Cc electrophoresis detection.

[0040] Figure 4 shows the results of C4P7Cd electrophoresis detection.

[0041] Figure 5 shows the results of C4P7Ce electrophoresis detection.

[0042] Figure 6 shows the results of C4P7Cf electrophoresis detection.

[0043] Figure 7 shows the results of C4P7Cg electrophoresis detection.

[0044] Figure 8 shows the results of C4P7Ch electrophoresis detection.

[0045] Figure 9 shows the results of C4P7Ea electrophoresis detection.

[0046] Figure 10 shows the electrophoresis detection results for C4P7Eb.

[0047] Figure 11 shows the results of C4P7Ec electrophoresis detection.

[0048] Figure 12 shows the effect of C4P7Ch collagen on cell adhesion.

[0049] Figure 13 shows the effect of C4P7Cf collagen on cell adhesion. Detailed Description

[0050] As used in this document, a “recombinant collagen” refers to an amino acid sequence encoded by a specific engineered or modified gene, or a fragment thereof, or a combination of fragments of such a functional amino acid sequence, which is prepared by recombinant DNA technology. The coding sequence Petition 870260016232, dated 20 / 02 / 2026, page 26 / 126 / 55. The gene or amino acid sequence of recombinant collagen may have low homology with the gene coding sequence or amino acid sequence of human collagen. A “recombinant humanized collagen” refers to the full-length or partial-length amino acid sequence fragment encoded by a specific human collagen type gene prepared by recombinant DNA technology, or a combination containing functional fragments of human collagen. In this document, recombinant collagen comprises one or more repeat units. The repeat unit may be derived from human type IV collagen. Thus, recombinant collagen may be recombinant humanized type IV collagen. The plurality of repeat units may be linked by means of a linker, which may be natural amino acid residue(s), for example, 1 to 50 amino acid residues, from the repeat units in human type IV collagen.A repeating unit can be SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33. A recombinant collagen can be SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32.

[0051] As used in this document, the term “variant” means a recombinant collagen having cell adhesion activity that includes alterations / mutations (i.e., substitutions, additions, insertions, and / or deletions) at one or more positions. Substitution means the exchange of an amino acid occupying a given position for a different amino acid; deletion means the removal of an amino acid occupying a given position; and insertion means the addition of an amino acid adjacent to and immediately following an amino acid occupying a given position. Addition refers to the addition of one or more amino acid residues to the C-terminus and / or N-terminus of an amino acid sequence. The substitution may be a conservative substitution. A variant of a repeating unit may be a sequence in which one or more amino acid residues in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33 are altered or mutated (i.e., Petition 870260016232, dated 20 / 02 / 2026, p. 27 / 126 / 55 substituted, added, inserted and / or deleted). A variant of recombinant collagen may be a sequence in which one or more amino acid residues in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32 are altered or mutated (i.e., substituted, added, inserted and / or deleted).

[0052] For example, a variant of a repeat unit of SEQ ID NO: 1 may be a variant comprising the following mutations: the addition of Gfpgfp (SEQ ID NO: 34) or the N-terminal truncated fragment of SEQ ID NO: 34 (e.g., 1 to 5 amino acid residues, such as 1, 2, 3, 4, or 5 amino acid residues are truncated from the N-terminal of SEQ ID NO: 34, corresponding to the residues fpgfp (SEQ ID NO: 58), pgfp (SEQ ID NO: 59), gfp, fp, and pp, respectively) to the N-terminal of the amino acid sequence of SEQ ID NO: 1, and / or the addition of GFMGPPGPQGQPGLP (SEQ ID NO: 35) or a C-terminal truncated fragment of SEQ ID NO: 35 (e.g., 1 to 14 amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues are truncated from the C-terminal of SEQ ID NO: 35 to the C-terminal of the amino acid sequence of SEQ ID NO: 1.A variant of a repeat unit of SEQ ID NO: 1 may be a variant comprising the following mutations: the addition of Gfpgfp (SEQ ID NO: 34) or the N-terminal truncated fragment thereof (e.g., 1, 2, 3, 4, or 5 amino acid residues are truncated from the N-terminal of SEQ ID NO: 34) to the N-terminal of the amino acid sequence of SEQ ID NO: 1 and / or truncation of 1 to 5 amino acid residues, such as 1, 2, or 3 amino acid residues, at the C-terminal of the amino acid sequence of SEQ ID NO: 1.

[0053] A variant of a repeat unit of SEQ ID NO: 28 may be a variant comprising the following mutations: the addition of Glpgtp (SEQ ID NO: 36) or the N-terminal truncated fragment thereof (e.g., 1 to 5 amino acid residues, e.g., 1, 2, 3, 4 or 5 residues). Petition 870260016232, dated 20 / 02 / 2026, p. 28 / 126 / 55 of amino acids are truncated from the N-terminal of SEQ ID NO: 36) to the N-terminal of the amino acid sequence of SEQ ID NO: 28.

[0054] In the context of the present description, a conservative substitution may be defined as a substitution within one or more of the amino acid classes reflected in one or more of the following tables: Conservative classes of amino acid residues:

[0055] Acid residues D and E

[0056] Basic waste K, R and H

[0057] Uncharged hydrophilic residues S, T, N and Q

[0058] Aliphatic uncharged residues G, A, V, L and I

[0059] Non-polar uncharged residues C, M and P

[0060] Aromatic residues F, Y and W. Alternative physical and functional classification of amino acid residues:

[0061] Waste containing alcohol groups S and T

[0062] Aliphatic residues I, L, V and M

[0063] Residues related to cycloalkenyl F, H, W and Y

[0064] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y

[0065] Negatively charged residues D and E

[0066] Polar residues C, D, E, H, K, N, Q, R, S and T

[0067] Positively charged residues H, K and R

[0068] Small waste A, C, D, G, N, P, S, T and V

[0069] Minimum waste A, G and S

[0070] Residues involved in the formation of turns A, C, D, E, G, H, K, N, Q, R, S, P and T

[0071] Flexible waste Q, T, K, S, G, P, D, E and R.

[0072] As used in this document, “cell adhesion” refers to the adhesion between cells and collagen. Collagen, such as the recombinant collagen described in this document, can facilitate adhesion between cells and the container in which the cells are grown. Petition 870260016232, dated 20 / 02 / 2026, p. 29 / 126 / 55

[0073] As used in this document, 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.

[0074] As used in this document, the term “expression vector” means a linear or circular DNA molecule comprising a polynucleotide encoding a recombinant collagen protein and operatively linked to a control sequence provided for its expression.

[0075] As used in this document, the term “host cell” means any type of cell capable of transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present description. The term “host cell” includes any progeny of a precursor cell that is not identical to the mother cell due to a mutation that occurs during replication.

[0076] As used in this document, the term “nucleic acid” means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or is modified in a way that is not present in nature to contain a segment of a nucleic acid, or is synthetic, and the nucleic acid molecule may comprise 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, for example, a nucleic acid that is codon-optimized for expression in E. coli cells.

[0077] The term “operatively linked” means a configuration in which a control sequence is placed in an appropriate position relative to a coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence. Petition 870260016232, dated 20 / 02 / 2026, page 30 / 126 / 55

[0078] The degree of association between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For the purposes of this description, the sequence identity between two amino acids is determined by the Needleman-Wunsch Algorithm implemented by the Needle program (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet. 16: 276-277) (Version 5.0.0 or later is preferred). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). Needle's output labeled "Longest Identity" (obtained using the non-simplified option) is used as the percentile identity and calculated as follows: (identical residues x 100) / (alignment length - total number of gaps in the alignment)

[0079] For the purposes of this description, the sequence identity between two deoxynucleotide sequences is determined by the Needleman-Wunsch Algorithm implemented by the Needle program (Needleman and Wunsch, 1970) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000) (Version 5.0.0 or later is preferred). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4). The Needle output labeled “Longest Identity” (obtained using the non-simplified option) is used as the percent identity and calculated as follows: (identical deoxyribonucleotides x 100) / (alignment length / total number of gaps in the alignment) Recombinant collagen Petition 870260016232, dated 20 / 02 / 2026, p. 31 / 126 / 55

[0080] The present description provides a recombinant collagen comprising one or more repeating units linked directly or via a linker, wherein the repeating units comprise an amino acid sequence selected from SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33, or a variant thereof. The variant may 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 with the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33. With respect to the recombinant collagen described in this document, the mutation may be selected from the group that consists of a replacement, addition, insertion, or deletion.Preferably, the substitution is a conservative amino acid substitution.

[0081] The recombinant collagen described in this document may comprise a plurality of repeating units, for example, 2 to 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 or 50 repeating units.

[0082] The linker in recombinant collagen described in this document may comprise one or more amino acid residues, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 amino acid residues.

[0083] Recombinant collagen is recombinant human type IV collagen or recombinant humanized type IV collagen, preferably having cell adhesion activity. The recombinant collagen described in this document may be derived from humans and is therefore recombinant humanized type IV collagen. Petition 870260016232, dated 20 / 02 / 2026, p. 32 / 126 / 55

[0084] The recombinant collagen described in this document may also comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32, or a variant thereof, wherein the variant is (1) an amino acid sequence having one or more amino acid residue mutations in said 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 said amino acid sequence. Nucleic acid constructs

[0085] The present description also refers to a nucleic acid construct comprising a nucleic acid of the present description operatively linked to one or more control sequences that direct the expression of a coding sequence in a suitable host cell under a condition compatible with the control sequences. The vector may comprise a nucleic acid construct.

[0086] Nucleic acids can be modified in a variety of ways to enable the expression of recombinant collagen. Depending on the expression vector, it may be desirable or necessary to modify 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.

[0087] The control sequence may be a promoter, that is, it is recognized by a host cell for the expression of a polynucleotide encoding a recombinant collagen of the present description. The promoter comprises transcriptional control sequences that mediate the expression of recombinant collagen. The promoter may be any nucleic acid that exhibits transcriptional activity in a host cell, including variants, truncated or hybrid promoters, and may be Petition 870260016232, dated 20 / 02 / 2026, page 33 / 126 / 55 obtained from a gene that encodes an extracellular or intracellular recombinant collagen homologous or heterologous to the host cell.

[0088] Examples of promoters suitable for directing transcription of the vectors or nucleic acid constructs described in bacterial host cells are promoters obtained from: Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltoamylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene, E. coli lac operon and E. coli trc promoter.

[0089] In yeast hosts, useful promoters are obtained from the following genes: 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.

[0090] The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator may be operatively linked to the 3' end of the polynucleotide encoding recombinant collagen. Any terminator that is functional in the host cell may be used in the present description.

[0091] Preferred terminators for bacterial host cells are obtained from the alkaline protease genes of Bacillus clausii (aprH), alpha-amylase of Bacillus licheniformis (amyL) and ribosomal RNA of E. coli (rrnB).

[0092] Preferred terminators for yeast host cells are obtained from the following genes: Saccharomyces enolase Petition 870260016232, dated 20 / 02 / 2026, page 34 / 126 / 55 cerevisiae, 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).

[0093] The control sequence can also be a stabilizing mRNA region downstream of the promoter and upstream of the gene's coding sequence, which increases gene expression.

[0094] Examples of suitable mRNA stabilizing regions are obtained from the following genes: cryllIIA gene from Bacillus thuringiensis (WO 94 / 25612) and SP82 gene from Bacillus subtilis (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

[0095] The control sequence can also be a leader sequence, that is, an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the polynucleotide encoding recombinant collagen. Any leader sequence that is functional in the host cell can be used.

[0096] Suitable leader sequences for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae factor alpha, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde 3-phosphate dehydrogenase (ADH2 / GAP).

[0097] The control sequence can also be a polyadenylation sequence, which is operatively linked to the 3' end of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0098] Useful polyadenylation sequences for host cells of Petition 870260016232, dated 20 / 02 / 2026, page 35 / 126 / 55 yeast are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0099] The control sequence may also be a signal peptide-coding region that encodes a signal peptide bound to the N-terminal of recombinant collagen and directs the recombinant collagen to the secretory pathway of the cell. The 5' end of the polynucleotide coding sequence may contain a signal peptide-coding sequence that is naturally bound in the open reading frame to the coding sequence segment that encodes recombinant collagen. Alternatively, the 5' end of the coding sequence may contain a signal peptide-coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide-coding sequence, a foreign signal peptide-coding sequence may be required.Alternatively, the foreign signal peptide coding sequence can simply replace the natural signal peptide coding sequence to increase the secretion of recombinant collagen. However, any signal peptide coding sequence that directs the expressed recombinant collagen to the host cell's secretory pathway can be used.

[00100] An effective signal peptide coding sequence for a bacterial host cell is a signal peptide coding sequence obtained from the following genes: maltogenic amylase from Bacillus NCIB 11837, subtilisin from Bacillus licheniformis, beta-lactamase from Bacillus licheniformis, alpha-amylase from Bacillus stearothermophilus, neutral protease from Bacillus stearothermophilus (nprT, nprS, nprM), and prsA from Bacillus subtilis. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[00101] Useful signaling peptides for yeast host cells are Petition 870260016232, dated 20 / 02 / 2026, page 36 / 126 / 55 obtained from the following genes: Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (Yeast 8: 423488). Expression vector

[00102] The description also refers to recombinant expression vectors comprising a nucleic acid, a promoter, and transcription and translation termination signals of the present description. Nucleic acids and control sequences can be ligated to produce recombinant expression vectors that may include one or more restriction sites convenient for insertion or substitution of a polynucleotide encoding recombinant collagen at such sites. Alternatively, the polynucleotide can be expressed by inserting a nucleic acid, or a nucleic acid construct comprising the nucleic acid, into an appropriate expression vector. When an expression vector is produced, the coding sequence is localized in the vector so that the coding sequence is operatively linked to an appropriate expression control sequence.

[00103] A recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to a recombinant DNA procedure and can allow the expression of a polynucleotide. The choice of vector will normally depend on the compatibility of the vector with the host cell into which the vector will be introduced. The vector can be a linear chain or a closed circular plasmid.

[00104] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The Petition 870260016232, dated 20 / 02 / 2026, page 37 / 126 / 55: A vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates with one or more chromosomes into which it has been integrated. Furthermore, separate vectors or plasmids may be used, or two or more vectors or plasmids that collectively contain the total DNA to be introduced into the host cell genome, or transposons may be used.

[00105] The vector preferably contains one or more selectable markers that allow convenient selection of transformed cells, transfected cells, transduced cells and the like. A selectable marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophy to auxotrophs, etc.

[00106] Examples of selectable bacterial markers are the dal gene of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance, such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline. Suitable markers for yeast host cells include, but are not limited to: ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[00107] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.

[00108] The vector may contain an element that allows the vector to integrate into the host cell genome or the vector to autonomously replicate in the cell independently of the genome.

[00109] For integration into the host cell genome, the vector may rely on the polynucleotide sequence that codes for recombinant collagen or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector Petition 870260016232, dated 20 / 02 / 2026, page 38 / 126 / 55 may contain an additional polynucleotide to direct homologous recombination integration into the host cell genome at precise location(s) on the chromosome(s). To increase the probability of integration at a precise location, the integrational element must contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have a high degree of sequence identity with the corresponding target sequences to increase the probability of homologous recombination. The integrational element may be any sequence homologous to a target sequence within the host cell genome. Furthermore, the integrational element may be a coding or non-coding polynucleotide. In another aspect, the vector can be integrated into the host cell's genome through non-homologous recombination.

[00110] To replicate autonomously, the vector may additionally comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator that functions in cells to mediate autonomous replication. The term “origin of replication” or “plasmid replicator” means a polynucleotide that allows a plasmid or vector to replicate in vivo.

[00111] Examples of bacterial replication origins are the pBR322, pUC19, pACYC177, and pACYC184 plasmid replication origins that allow replication in E. coli, as well as the pUB110, pE194, pTA1060, and pAMe1 plasmid replication origins that allow replication in Bacillus.

[00112] Examples of origins of replication for use in yeast host cells are the 2-micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, as well as the combination of ARS4 and CEN6. Petition 870260016232, dated 20 / 02 / 2026, page 39 / 126 / 55

[00113] More than one copy of the polynucleotide of the present description can be inserted into a host cell to increase the production of recombinant collagen. A greater number of copies of a 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 along with the polynucleotide, wherein cells comprising amplified copies of the selectable marker gene and therefore additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selectable agent.

[00114] The procedures for linking the elements described above to construct the recombinant expression vectors of the present description 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)). Host cell

[00115] The present description also refers to recombinant host cells comprising a polynucleotide of the present description operatively linked to one or more control sequences that direct the production of the recombinant collagen of the present description. The construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integral or as an extrachromosomal vector for autonomous replication, as described above. The term “host cell” encompasses any progeny of a precursor cell that is not identical to the mother cell due to a mutation that occurs during replication. The choice of host cell will depend largely on the gene encoding the recombinant collagen and its source.

[00116] The host cell can be any cell useful in the recombinant production of the recombinant collagen of the present description, for example, Petition 870260016232, dated 20 / 02 / 2026, p. 40 / 126 / 55 a prokaryote or a eukaryote.

[00117] The prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. 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, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[00118] The host cell can also be a cell from a eukaryotic organism, such as a mammal, insect, plant, or fungus. Plant cells in this document do not include plant cells that can regenerate into an entire plant. Animal cells also do not include cells that can generate an animal body.

[00119] Host cells can be fungal cells, such as Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota, among others. The host cell can be a yeast cell, including ascosporogenic yeast (Endomycetales), basidiosporogenic yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). The yeast host cell can be a cell of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as a cell of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica. Production Method

[00120] The present description also refers to a method of producing recombinant collagen as described in this document, comprising: (1) incubate the host cell described in this document under Petition 870260016232, dated 20 / 02 / 2026, page 41 / 126 / 55 an adequate cultural condition; (2) collect the host cell and / or culture medium comprising recombinant collagen; and (3) purify the recombinant collagen.

[00121] Using methods known in the art, host cells are cultured in a suitable nutrient medium for the production of recombinant collagen. For example, cells can be cultured in shake flasks or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, where cultivation is carried out in a suitable medium and under conditions that allow the expression and / or isolation of recombinant collagen. Cultivation is carried out in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media can be obtained from commercial suppliers or can be prepared according to compositions described (e.g., in the catalogs of the American Type Culture Collection).If recombinant collagen is secreted into the nutrient medium, the recombinant collagen can be recovered directly from the medium. If recombinant collagen is not secreted, it can be recovered from the cell lysate.

[00122] 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, an adhesion assay can be used to determine the activity of recombinant collagen.

[00123] Recombinant collagen can be recovered by 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, drying by Petition 870260016232, dated 20 / 02 / 2026, page 42 / 126 / 55 pulverization, evaporation or precipitation. In one aspect, recombinant collagen is recovered from the fermentation broth comprising recombinant collagen.

[00124] Recombinant collagen can be purified by 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 electrophoresis), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure recombinant collagen.

[00125] Step (1) may comprise one or more of the following steps: construction of an expression plasmid, for example, insertion of a coding nucleotide sequence into a pET28a-Trx-His expression vector to obtain a recombinant expression plasmid. Successfully constructed expression plasmids may be transformed into E. coli cells (e.g., competent E. coli BL21 (DE3) cells). The specific process may be as follows: (1) harvest the plasmid to be transformed and add it to E. coli cells.competent coli BL21 (DE3); (2) place the mixture on ice in an ice bath (e.g., for 10 to 60 min, e.g., 30 min), followed by a thermal shock in a water bath (e.g., at 40 to 50°C, e.g., 42°C, for 45 to 90 s), remove the mixture and place it on ice in an ice bath (e.g., for 1 to 5 min, e.g., 2 min); (3) add liquid LB medium followed by incubation (e.g., at 35 to 40°C, e.g., 37°C, at 150 to 300 rpm, e.g., 220 rpm for 40 to 80 min, e.g., 60 min); (4) spread the bacterial solution and collect individual colonies. For example, the bacterial solution is collected and spread evenly on LB plates containing ampicillin sodium, and the plates are cultured in an incubator at 37°C for 15 to 17 hours until colonies of... Petition 870260016232, dated 20 / 02 / 2026, page 43 / 126 / 55 uniform size grow on the plates.

[00126] Step (2) may involve culturing individual colonies in LB medium containing an antibiotic stock (e.g., in a shaker at 150 to 300 rpm, e.g., 220 rpm, at a constant temperature of 35 to 40°C, e.g., 37°C for 5 to 10 hours, e.g., 7 hours). Then the cultured shaker flask is cooled to 10 to 20°C, e.g., 16°C, and IPTG is added to induce expression for a period of time, and then the cells are collected (e.g., by centrifugation).

[00127] Step (3) may include resuspending the bacterial cells with an equilibrium working solution, cooling the bacterial liquid to < 15°C, performing homogenization (e.g., high-pressure homogenization, e.g., 1 to 5 times, e.g., 2 times) and separating the homogenized bacterial liquid to obtain a supernatant. The equilibrium working solution may comprise 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, at pH 7 to 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 Tris concentration can be 10, 15, 20, 25, 30, 35, 40, 45 or 50 nM. The imidazole concentration 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.

[00128] Step (3) may include purification and enzymatic digestion of recombinant collagen. Purification may be a crude purification comprising a Ni-agarose column purification of the supernatant to obtain an eluate containing the target protein. The crude purification may comprise washing the column material with water, for example, to 2 to 10 column volumes (CVs), for example, 5 CVs. The column material may be equilibrated with an equilibration solution (200 mM chloride of Petition 870260016232, dated 20 / 02 / 2026, p. 44 / 126 / 55 sodium, 25 mM Tris, 20 mM imidazole at pH 8.0), for example, for 2 to 10 CVs, for example, 5 CVs. The equilibrium solution may comprise 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole at pH 7 to 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, 440, 450, 460, 470, 480 or 490 nM. The Tris concentration can be 10, 15, 20, 25, 30, 35, 40, 45 or 50 nM. The imidazole concentration 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.

[00129] Step (3) may involve loading the supernatant into the column material and washing off impurity proteins with a washing solution.The washing solution may include 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole at pH 7 to 9. For example, the sodium chloride concentration 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 Tris concentration can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The imidazole concentration 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, the eluent can be added and the continuous flow liquid is collected. The eluent can comprise 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 100 to 500 mM imidazole at 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. Petition 870260016232, dated 20 / 02 / 2026, p. 45 / 126 / 55 470, 480 or 490 nM. The pH can be 7, 7.5, 8, 8.5 or 9.

[00130] Enzymatic digestion may involve the addition of a collagen processing enzyme for enzymatic digestion (in a ratio of total protein to total collagen processing enzyme of 10 to 100:1, e.g., 50:1, at 10 to 20°C, e.g., 16°C for 2 to 8 hours, e.g., 4 hours). The enzymatically digested protein solution is dialyzed, e.g., in a dialysis bag at 1 to 6°C, e.g., 4°C for 1 to 8 hours, e.g., 2 hours, and then transferred to a new dialysate for dialysis at 1 to 6°C, e.g., 4°C overnight.

[00131] Purification may include fine purification (e.g., for proteins with an isoelectric point > 8.0). Preferably, fine purification comprises the use of a strong anion-exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). The eluate containing the target protein or the product after enzymatic digestion (e.g., enzymatically digested and dialyzed product) is subjected to gradient elution. Gradient elution comprises 0 to 15% solution B for 1 to 5 minutes, followed by retention for 1 to 5 column volumes, e.g., 3 column volumes; 15 to 30% solution B for 1 to 5 minutes, followed by retention for 1 to 5 column volumes, e.g., 3 column volumes; 30 to 50% solution B for 1 to 5 minutes, followed by retention for 1 to 5 column volumes, e.g., 3 column volumes; 50 to 100% solution B for 1 to 5 minutes, followed by retention for 1 to 5 column volumes, e.g., 3 column volumes.Solution B may comprise 10 to 50 mM Tris, 0.5 to 5 M sodium chloride, at pH 7 to 9. For example, the Tris concentration is 15, 20, 25, 30, 35, 40, or 45 mM. The sodium chloride concentration is 1, 2, 3, or 4 M. The pH may be 7, 7.5, 8, 8.5, or 9. Fine purification may include equilibrizing the column material with solution A and loading, followed by gradient elution. Solution A may contain 10 to 50 mM Tris, 10 to 50 mM of... Petition 870260016232, dated 20 / 02 / 2026, p. 46 / 126 / 55 sodium chloride, at pH 7 to 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.

[00132] To clarify the purpose, technical solutions, and advantages of the present description, the technical solutions in the examples will be described clearly and completely below, in conjunction with the following examples. Obviously, the examples described are partial embodiments of the present description, and not all embodiments. Based on the examples in the present description, all other embodiments obtained by those skilled in the art without doing any creative work fall within the scope of protection of the present description.

[00133] The following examples are provided additionally to illustrate the present description. Examples

[00134] The present description is further illustrated by the following examples, but any example or combination thereof shall not be construed as limiting the scope of the present description. The scope of the present description is defined by the following claims. As one skilled in the art combines this descriptive report with common knowledge of the art, he can clearly discern the scope defined by the claims. Without departing from the spirit and scope of the present description, those skilled in the art may make modifications to the technical solution of the present description. Such modifications also fall within the scope of the present description. Example 1: Construction, Expression, and Screening of Recombinant Humanized Type IV Collagen Fragments

[00135] 1. A large-scale screening of functional regions was performed to obtain the following functional regions of different target genes from recombinant humanized type IV collagen. Petition 870260016232, dated 20 / 02 / 2026, page 47 / 126 / 55

[00136] 1) The amino acid sequence of C4P7Ch (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeq, SEQ ID NO: 1, and the number of repeating units is 10; the amino acid sequence of C4P7Ch is SEQ ID NO: 2): Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq (SEQ ID NO: 2) Base sequence C4P7Ch (SEQ ID NO: 3): GGGGCTAAAGGAGACAAGGGCAGCAAGGGCGAAGT CGGTTTCCCAGGTCTGGCTGGTAGCCCGGGCATCCCGGGTTCAAAG GGTGAACAAGGTGCTAAAGGCGACAAAGGCAGCAAGGGTGAGGT TGGTTTCCCGGGTCTGGCGGTTCTCCAGGCATCCAGGTCGAAA GGAGAACAAGGTGCGAAAGGGCGATAAAGGCTCCAAGGGTGAAGT GGGCTTCCCGGGTTTAGCCGGTAGCCCAGGTATTCCGGGTAGCAA AGGCGAACAGGGTGCGAAAGGCGACAAAGGGAGTAAGGGCGAGG GGGCGAACAGGGCGCGAAAGGTGATAAAGGCAGCAAAGGCGAGG TTGGCTTCCCGGGTCTGGCAGGTAGCCCGGGTATCCCGGGTAGCA AGGGTGAGCAGGGTGCCAAAGGCGACAAAGGTAGCAAGGGGGAA GTGGGTTTTCCGGGACTGGAGGTAGGCCCGGGTTTTA AGGGCGAGCAGGGTGCGAAAGGTGACAAAGGTAGCAAGGGCGAG GTTGGCTTTCCGGGCTTGGCGGGTAGCCCGGGCATTCCGGGCTCCA Petition 870260016232, of 20 / 02 / 2026, p. 48 / 126 / 55 AGGGTGAACAAGGTGCGAAAGGTGATAAAGGCTCTAAGGGTGAG GTTGGTTTTCCGGGTCTGGCGGGTTCCCCGGGCATTCCGGGCTCGA AGGGCGAGCAAGGTGCTAAAGGTGATAAGGGCTCCAAGGGGCGAG AGGGCGAACAGGGTGCGAAAGGCGATAAAGGTTCCAAGGGCGAA GTCGGATTCCCTGGCCTCGCCGGTAGCCCGGGCATCCCTGGCTCCA AGGGCGAGCAG

[00137] 2) C4P7Cf (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, SEQ ID NO: 4, and the number of repeating units is 8; the amino acid sequence of C4P7Cf is SEQ ID NO: 5): Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq (SEQ ID NO: 5) Base sequence C4P7Cf (SEQ ID NO: 6): GGAGCTAAAGGGGACAAAGGCTCCAAAGGCGAAGTC GGTTTCCCGGGCCTGGCGGTAGCCCGGGTATCCCGGGTAGCAAG GGTGAGCAGGGGTTCATGGGTCCACCGGGCCCAGGGTGCCAAA GGTGATAAAGGTTCTAAGGCGAGGTGGGTTTCCCG GGTTCTCCGGGCATTCCGGGAAGCAAGGGTGAACAGGGCTTTATG GGTCCGCCAGGTCCGCAGGGTGCGAAAGGTGATAAAGGCAGCAA GGGAGAAGTTGGCTTCCCGGGCCTGGCAGGCAGCCCGGGCATTCC GGGGTCGAAGGGCGAACAAGGTTTCATGGGTCCGCCGA Petition 870260016232, of 20 / 02 / 2026, p. 49 / 126 / 55 AGGTGCGAAAGGTGATAAGGGTAGCAAGGGTGAAGTGGGTTTTCC GGGATTAGCGGGTTCTCCGGGCATTCCGGGTTCAAAAGGTGAACA AGGCTTTATGGGTCCGCCTGGCCCAGGGTGCTAAAGGCGACAA GGGTAGCAAAGGCGAGGTAG GGGCATTCCGGGTTCCAAGGGCGAGCAGGGTTTTATGGGCCCACC GGGCCCGCAAGGCGCAAAAGGTGATAAGGGCAGCAAAGGCGAGG TGGGCTTCCCGGGACTGGCAGGTTCTCCGGGTATCCCGGGTTCCAAA AGGCGACAAAGGTAGCAAGGGCGAAGTTGGTTTTCCGGGCCTGGC TGGTTCGCCGGGCATCCCGGGCTCCAAGGGCGAGCAAGGCTTCAT GGGTCCACCGGGTCCGCAAGGTGCCAAAGGCGACAAAGGTAGCA GGGGTCCAAGGGTGAACAGGGCTTTATGGGTCCGCCGGGCCCTCA A

[00138] 3) Amino acid sequence C4P7Ca (the amino acid sequence of the repeat unit is GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQ PGLP, SEQ ID NO: 7, and the number of repeat units is 6 amino acid sequences; of C4P7Ca is SEQ ID NO: 8): GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF MGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF MGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF MGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF MGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF MGPPGPQGQPGLP Petition 870260016232, dated 20 / 02 / 2026, page 50 / 126 / 55 GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF MGPPGPQGQPGLP (SEQ ID NO: 8) C4P7Ca base sequence (SEQ ID NO: 9): GGATTTCCCGGGTTCCCGGGTGCCAAAGGGGATAAA GGTTCAAAGGGCGAAGTGGGTTTCCCGGGTTTGGCTGGTAGCCCG GGTATCCCGGGTAGCAAAGGCGAACAGGGCTTTATGGGTCCGCCA GGACCGCAGGGTCAACCGGGACTGCCGGGTTTTCCGGGCTTCCCG GGTGCGAAAGGCGATAAAGGTTCCAAGGGTGAAGTTGGTTTTCCG GGTCTTGCAGGCAGCCCGGGTATTCCGGGTTCCAAGGGTGAACAG GGTTTCATGGGTCCACCGGGCCCACAAGGTCAGCCGGGTCTGCCT GGTTTCCCGGGCTTCCCGGGTGCCAAAGGCGACAAAGGTAGCAAG GGCGAAGTTGGCTTTCCGGGTCTGGCGGGTTCGCCGGGCATTCCGG GCTCGAAGGGCGAGCAGGGTTTCATGGGCCCACCGGGTCCGCAGG GTCAGCCTGGCCTGCCGGGATTCCCAGGTTTTCCGGGAGCGAAAG GCGACAAGGGTAGTAAGGGTGAGGTCGGTTTTCCAGGCTTGGCGG GCTCTCCCGGTATCCCGGGCTCTAAGGGCGAGCAAGGCTTTATGG GTCCACCGGGTCCGCAAGGTCAACCTGGATTACCGGGATTCCCAG GCTTTCCGGGCGCGAAAGGCGATAAAGGCAGCAAGGGTGAGGTG GGCTTCCCGGGCCTCGCGGGTAGCCCGGGCATCCCGGGTAGCAAG GGTGAGCAGGGCTTCATGGGTCCTCCGGGTCCGCAGGGCCAACCG GGCCTGCCGGGATTCCCGGGTTTCCCGGGCGCTAAAGGCGACAAA GGCAGCAAGGGTGAGGTTGGTTTTCCGGGTCTGGCAGGTAGCCCG GGCATTCCGGGCTCCAAGGGCGAACAGGGTTTTATGGGTCCACCG GGCCCTCAAGGTCAGCCGGGCCTGCCG

[00139] 4) Amino acid sequence C4P7Cb (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, SEQ ID NO: 10, and the number of repeating units is 8; the amino acid sequence of C4P7Cb is SEQ ID NO: 11): Petition 870260016232, dated 20 / 02 / 2026, page 51 / 126 / 55 Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq (SEQ ID NO: 11) Base sequence C4P7Cb (SEQ ID NO: 12): GGATTTCCCGGGTTTCCGGGTGCGAAAGGCGACAAG GGTTCCAAGGGTGAAGTTGGTTTTCCGGGGCTCGCTGGTAGCCCGG GCATCCCGGGTAGCAAGGGCGAACAGGGTTTCATGGGCCCACCGG GCCCGCAGGGTTTTCCGGGTTTCCCGGGCGCGAAAGGCGACAAAG GCAGCAAGGGTGAAGTGGGCTTTCCGGGCCTGGCGGGTAGTCCGG GCATCCCGGGCTCGAAGGGCGAACAGGGCTTTATGGGCCCACCAG GCCCACAGGGCTTCCCGGGATTCCCGGGTGCCAAAGGCGATAAAG GTTCAAAGGGCGAAGTCGGTTTCCCGGGTCTGGCAGGCTCTCCGG GCATCCCGGGCTCTAAGGGTGAACAAGGTTTCATGGGTCCACCGG GCCCGCAGGGCTTTCCGGGTTTTCCGGGTGCGAAAGGCGACAAGG GCAGCAAAGGTGAGGTTGGCTTTCCGGGCTTGGCGGGTAGCCCGG GTATCCCGGGTAGCAAGGGTGAGCAGGGTTTCATGGGTCCGCCTG GACCGCAAGGTTTCCCGGGTTTCCCGGGTGCGAAAGGTGATAAAG GTAGCAAGGGCGAGGTAGGTTTTCCGGGTCTGGCAGGCTCCCCGG GCATTCCGGGCTCTAAGGGTGAGCAGGGTTTCATGGGTCCGCCAG GTCCGCAAGGTTTCCCGGGATTTCCGGGTGCTAAAGGTGATAAAG GTTCCAAAGGTGAAGTGGGTTTTCCAGGCCTGGCCGGCAGCCCGG GCATTCCGGGCTCCAAGGGCGAGCAGGGCTTCATGGGTCCGCCTG GCCCTCAAGGCTTCCCCGGCTTCCCGGGAGCTAAGGGCGACAAAG Petição 870260016232, de 20 / 02 / 2026, pág. 52 / 126 / 55 GTTCCAAGGGCGAGGTGGGCTTCCCGGGCTTGGCGGGTAGCCCGG GCATTCCGGGTAGCAAGGGTGAGCAAGGCTTTATGGGTCCGCCAG GTCCGCAAGGGTTCCCGGGATTCCCGGGTGCGAAAGGTGATAAAG GCTCGAAGGGTGAGGTTGGTTTTCCGGGTCTGGCAGGTAGCCCGG GGATTCCGGGTAGCAAAGGCGAACAAGGTTTCATGGGTCCTCCGG GTCCACAG

[00140] 5) C4P7Cc amino acid sequence (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm, SEQ ID NO: 13, and the number of repeating units is 8; the amino acid sequence of C4P7Cc is SEQ ID NO: 14): Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm (SEQ ID NO: 14) Base sequence C4P7Cc (SEQ ID NO: 15): GGATTTCCCGGGTTCCCGGGTGCGAAAGGTGATAAA GGCAGCAAGGGTGAAGTCGGTTTTCCGGGTCTGGCAGGCAGCCCG GGTATCCCGGGTAGCAAAGGCGAACAGGGCTTTATGGGTTTCCCG GGCTTCCCAGGTGCGAAGGGCGATAAAGGTTCGAAAGGTGAGGTA GGTTTCCCGGGTTTAGCAGGTTCCCCGGGCATTCCGGGCAGCAAG GGTGAACAGGGTTTCATGGGCTTTCCGGGCTTCCCAGGCTAAA GGCGACAAAGGTTCTAAGGGTGAAGTGGGCTTCCCGGGTCTGGCT GGTAGCCCGGGCATCCCGGGCTCCAAGGGTGAGTTGGGGGG Petition 870260016232, of 20 / 02 / 2026, p. 53 / 126 / 55 GGTTTTCCGGGCTTCCCAGGCGCGAAAGGCGACAAAAGGCAGCAAG GGCGAGGTGGGTTTTCCGGGTTTGGCGGGTAGCCCGGGTATTCCG GGTTCGAAGGGTGAACAAGGTTTCATGGGTTTTCCGGGATTCCCAG GCGCGAAAGGCGATAAGGGCAGCAAGGGCGAGGTTTCCGTT GGACTGGCCGGAAGCCCGGGTATCCCGGGATCTAAGGGCGAACAA GGCTTTATGGGTTTCCCGGGTTTTCCTGGTGCGAAAGGCGATAAAG GCTCCAAGGGCGAGGTTGGTTTTCCAGGCCTGGCTGGCTCTCCGG CATTCCGGGTTAAGGGTGAGCAGGGGTTTCTT TTCCCGGGTGCAAAGGGTGACAAAGGTAGCAAGGGTGAAGTTGGC TTTCCGGGTCTGGCGGGTTCCCCGGGCATTCCGGGTAGCAAAGGTG AGCAAGGTTTTATGGGTTTTCCGGGCTTCCCGGGTGCCAAAGGCGA CAAAGGTAGCAAGGGAGAGGTGGTTGGCCGGGGGGGGGGGG CCCGGGCATCCCGGGCTCAAAGGGTGAACAGGGTTTCATG

[00141] 6) Amino acid sequence C4P7Cd (the amino acid sequence of the repeat unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeq, SEQ ID NO: 16, the number of repeat units is equivalent to 10 amino acid sequences; C4P7Cd is SEQ ID NO: 17): Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq (SEQ ID NO: 17) Base sequence C4P7Cd (SEQ ID NO: 18): Petition 870260016232, dated 20 / 02 / 2026, page 54 / 126 / 55 GGATTTCCCGGGTTCCCAGGTGCCAAGGGTGATAAA GGCAGCAAGGGCGAGGTGGGCTTCCCGGGCCTGGCTGGTTCACCG GGCATTCCGGGCAGTAAGGGTGAACAGGGTTTTCCGGGTTTCCCC GGCGCTAAAGGCGACAAGGGTAGCAAGGGGGAAGTAGGCTTCCC GGGTTTAGCTGGCTCTCCGGGCATTCCGGGCTCCAAGGGCGAACA GGGTTTCCCGGGCTTTCCGGGAGCGAAAGGCGACAAAGGCAGCAA GGGCGAAGTTGGTTTCCCGGGTCTGGCAGGTTCTCCGGGTATCCCG GGTAGCAAAGGCGAGCAGGGTTTCCCCGGCTTCCCGGGTGCGAAA GGTGATAAAGGTAGCAAGGGTGAGGTGGGTTTCCCTGGCCTGGCC GGTTCTCCGGGCATCCCGGGTTCTAAGGGAGAACAAGGTTTTCCG GGTTTTCCGGGTGCGAAAGGCGACAAAGGGTCCAAGGGTGAGGTT GGTTTTCCGGGTCTGGCGGGTTCACCGGGCATCCCGGGTAGCAAG GGCGAACAAGGCTTTCCAGGCTTCCCGGGTGCGAAAGGTGACAAA GGTTCCAAGGGCGAAGTTGGTTTTCCGGGTTTGGCGGGTAGCCCG GGTATCCCGGGTTCCAAGGGTGAGCAAGGTTTTCCGGGATTCCCG GGTGCCAAAGGTGACAAAGGTTCGAAGGGTGAAGTGGGTTTTCCA GGCTTGGCGGGTAGCCCGGGTATCCCGGGTTCCAAGGGTGAGCAG GGCTTCCCGGGTTTCCCGGGTGCAAAAGGTGATAAAGGCTCCAAA GGTGAGGTGGGCTTCCCTGGTCTCGCGGGTAGCCCGGGCATCCCG GGTAGCAAAGGTGAGCAGGGTTTCCCGGGCTTTCCAGGAGCTAAA GGGGATAAAGGCAGCAAGGGCGAGGTTGGCTTCCCGGGTCTGGCGGGTTCGCCGGGCATTCCGGGCAGCAAAGGCGAACAGGGCTTTCCG GGTTTTCCGGGTGCAAAGGGTGATAAAGGTAGCAAGGGCGAGGTC GGTTTTCCGGGCCTGGCAGGTAGCCCAGGCATTCCGGGTTCGAAG GGCGAACAA

[00142] 7) Amino acid sequence C4P7Ce (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp, SEQ ID NO: 19, and the number of repeating units is 8; the amino acid sequence of C4P7Ce Petition 870260016232, dated 20 / 02 / 2026, p. 55 / 126 / 55 is SEQ ID NO: 20): Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp (SEQ ID NO: 20) Base sequence C4P7Ce (SEQ ID NO: 21): GGGGCTAAAGGAGACAAGGGCAGCAAGGGCGAGGT TGGTTTCCCGGGCTTGGCGGGTTCTCCGGGTATCCCGGGCTCCAAG GGTGAACAGGGTTTTATGGGTCCGCCAGGTCCGCAGGGCCAACCT GGTCTGCCGGGCGCGAAAGGTGACAAAGGTAGCAAGGGCGAAGT TGGTTTTCCGGGCCTGGCCGGCAGCCCGGGCATTCCGGGCTCCAAA GGCGAGCAAGGCTTCATGGGTCCGCCTGGCCCACAGGGTCAACCG GGTCTGCCGGGCGCAAAAGGCGATAAAGGCAGCAAGGGCGAAGT AGGTTTCCCGGGCCTTGCTGGCTCTCCGGGTATTCCGGGTAGCAAG GGTGAACAGGGTTTTATGGGCCCACCGGGACCACAGGGTCAACCG GGTCTGCCGGGTGCTAAAGGTGATAAAGGCAGCAAGGGCGAGGTT GGTTTTCCGGGATTGGCGGGTTCTCCGGGCATCCCGGGTTCCAAGG GCGAACAAGGTTTCATGGGACCGCCTGGCCCACAGGGTCAACCGG GTCTGCCTGGTGCAAAAGGCGACAAAGGTAGCAAAGGCGAGGTCG GTTTTCCGGGCCTCGCGGGTAGCCCGGGAATCCCGGGTAGTAAGG GCGAACAGGGTTTCATGGGTCCACCGGGCCCACAGGGTCAGCCGG GTTTACCGGGTGCCAAAGGCGATAAAGGGTCGAAGGGCGAGGTGG GCTTCCCGGGTCTGGCAGGCAGCCCGGGTATTCCGGGCTCCAAGG GTGAGCAGGGTTTTATGGGTCCACCGGGACCGCAAGGTCAACCGG Petição 870260016232, de 20 / 02 / 2026, pág. 56 / 126 / 55 GTCTGCCGGGCGCTAAAGGCGATAAGGGTAGCAAAGGCGAGGTG GGCTTCCCGGGTCTGGCGGGTAGCCCGGGCATTCCGGGTTCCAAG GGTGAGCAGGGTTTCATGGGTCCGCCTGGTCCTCAAGGCCAGCCG GGGCTGCCCGGCGCGAAAGGTGACAAAGGTAGCAAGGGCGAAGT GGGTTTTCCGGGTTTGGCGGGTTCACCGGGCATCCCGGGCTCGAAG GGTGAACAGGGTTCATGGGTCCTCCGGGCCCGCAGGGACAACCG GGACTGCCG

[00143] 8) Amino acid sequence C4P7Cg (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfm, SEQ ID NO: 22, and the number of repeating units is 10; the amino acid sequence of C4P7Cg is SEQ ID NO: 23): Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm (SEQ ID NO: 23) Base sequence C4P7Cg (SEQ ID NO: 24): GGGGCTAAAGGAGACAAAGGTTCGAAAGGCGAGGT GGGCTTCCCAGGTCTGGCCGGTTCCCCGGGCATTCCGGGTAGCAA AGGCGAACAAGGTTTCATGGGTGCTAAAGGCGATAAAGGTAGCAA GGGTGAGGTTGGCTTCCCAGGCCTGGCTGGTTCCGTCCGTCCG GGCTCTAAGGGTGAACAAGGTTTCATGGGTGCAAAAGGTGATAAG GGTAGCAAGGGAGAAGTCGGTTTTCCGGGATTGGCGGGTAGCCCG Petition 870260016232, of 20 / 02 / 2026, p. 57 / 126 / 55 GGTATCCCGGGCAGCAAGGGCGAGCAGGGTTTTATGGGTGCAAAG GGCGACAAAGGTAGCAAGGGTGAGGTGGGCTTTCCGGGCCTCGCG GGTAGCCCTGGCATCCCGGGTTCCAAAGGTGAGCAAGGCTTCATG GGTGCTAAAGGTGATAAAGGCTCCAAAGGTGAAGTGGGTTTTCCG GGCCTGGCGGGTAGCCCGGGCATTCCGGGAAGCAAGGGCGAACA GGGTTTTATGGGCGCGAAGGGTGATAAAGGTAGTAAGGGCGAAGT TGGTTTCCCGGGCCTGGCTGGCTCTCCGGGTATCCCGGGCTCCAAA GGCGAGCAGGGTTTCATGGGTGCGAAAGGTGACAAGGGTAGCAA GGGTGAGGTGGGTTTCCCAGGTTTGGCGGGTAGCCCGGGCATTCC GGGTAGCAAGGGTGAACAAGGTTTCATGGGTGCGAAAGGTGACAA AGGCAGCAAGGGCGAGGTTGGTTTCCCGGGTCTGGCGGGTAGCCC GGGCATCCCGGGCTCTAAGGGCGAGCAGGGTTTTATGGGTGCCAA AGGCGACAAGGGCTCAAAGGGTGAAGTCGGTTTTCCGGGTTTAGC CGGTTCCCCGGGCATCCCGGGTTCTAAGGGTGAACAGGGCTTCAT GGGCGCGAAAGGAGATAAAGGCAGCAAAGGGGAAGTTGGTTTTC CAGGCCTGGCAGGCTCGCCGGGTATCCCGGGTTCCAAGGGCGAGC AGGGTTTTATG

[00144] 9) Amino acid sequence C4P7Ea (the amino acid sequence of the repeating unit is Glpgtpgptgpagqkgepgsdgipgsagekgepglp, SEQ ID NO: 25, and the number of repeating units is 10; the amino acid sequence of C4P7Ea is SEQ ID NO: 26): Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Petition 870260016232, dated 20 / 02 / 2026, p. 58 / 126 / 55 Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp (SEQ ID NO: 26) Base sequence C4P7Ea (SEQ ID NO: 27): GGACTACCCGGGACTCCGGGCCCAACCGGCCCAGCA GGCCAAAAGGGTGAACCGGGGTCCGACGGCATTCCGGGCAGCGCA GGTGAGAAAGGCGAACCGGGCTTGCCGGGGTTGCCGGGAACCCCG GGTCCGACCGGTCCAGCTGGCCAGAAAGGTGAGCCAGGAAGCGAT GGCATCCCGGGCTCGGCCGGTGAAAAAGGCGAGCCGGGTCTCCCG GGTCTGCCGGGAACCCCGGGTCCGACAGGCCCGGCTGGTCAGAAG GGCGAGCCGGGTAGCGACGGCATCCCGGGCAGCGCTGGTGAAAA AGGTGAGCCCGGTCTGCCGGGCTTGCCCGGCACCCCGGGACCGAC GGGCCCAGCAGGCCAGAAGGGCGAACCGGGTTCGGATGGTATTCC GGGCTCTGCCGGTGAGAAAGGCGAGCCCGGCTTGCCAGGCCTGCC TGGCACCCCGGGTCCGACCGGTCCGGCGGGCCAAAAAGGCGAGCC GGGTAGCGATGGTATCCCGGGCTCAGCCGGTGAGAAGGGTGAGCC GGGCCTGCCGGGCCTGCCTGGTACGCCGGGTCCGACCGGTCCGGC GGGCCAAAAGGGTGAACCGGGCTCCGACGGCATTCCGGGTTCTGC GGGTGAGAAAGGTGAACCGGGCCTGCCGGGCCTGCCCGGCACCCC GGGTCCTACGGGTCCGGCTGGTCAGAAAGGCGAGCCGGGCTCCGA TGGCATTCCGGGTTCTGCGGGTGAGAAGGGTGAACCGGGCTTGCC AGGTCTGCCGGGCACCCCGGGTCCGACGGGTCCGGCGGGTCAGAA GGGTGAGCCGGGTTCCGATGGTATCCCGGGCAGCGCGGGAGAAAA AGGTGAACCGGGTCTGCCGGGTCTTCCGGGTACTCCGGGTCCGAC CGGCCCTGCGGGTCAGAAGGGTGAGCCGGGAAGCGACGGCATCCC GGGCAGCGCGGGGGAGAAGGGTGAACCGGGTTTACCTGGCCTGCCGGGAACCCCGGGCCCTACCGGTCCGGCGGGTCAAAAGGGCGAACC GGGCAGCGACGGTATCCCGGGTAGCGCAGGCGAAAAAGGTGAAC CGGGCCTGCCG Petition 870260016232, dated 20 / 02 / 2026, page 59 / 126 / 55

[00145] 10) Amino acid sequence C4P7Eb (the amino acid sequence of the repeating unit is Gptgpagqkgepgsdgipgsagekgepglp, SEQ ID NO: 28, and the number of repeating units is 10; the amino acid sequence of C4P7Eb is SEQ ID NO: 29): Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp (SEQ ID NO: 29) Base sequence C4P7Eb (SEQ ID NO: 30): GGTCCCACAGGACCGGCAGGCCAGAAAGGTGAGCCG GGTTCCGACGGCATCCCGGGTTCGGCGGGTGAGAAAGGCGAGCCG GGTTTACCGGGTCCGACCGGTCCCGCGGGTCAAAAGGGCGAGCCG GGTAGCGATGGCATTCCGGGTTCTGCGGGTGAAAAGGGCGAACCG GGCCTCCCGGGTCCTACCGGTCCGGCGGGTCAGAAAGGCGAACCG GGCAGCGATGGCATCCCGGGCAGCGCGGGCGAGAAAGGCGAACC GGGCCTGCCGGGCCCGACCGGACCAGCTGGGCAAAAAGGTGAACC GGGCAGCGACGGCATCCCGGGTTCTGCAGGCGAGAAAGGTGAACC AGGCCTGCCGGGACCGACCGGTCCGGCAGGCCAGAAAGGTGAGCC TGGCAGTGATGGTATTCCGGGTTCTGCCGGTGAAAAAGGTGAGCC GGGCCTGCCGGGGCCAACGGGCCCAGCCGGACAAAAAGGTGAGC CGGGTTCCGACGGCATCCCGGGCTCCGCCGGTGAAAAGGGTGAGC CGGGCCTGCCTGGCCCAACGGGTCCGGCTGGCCAAAAGGGCGAGC CGGGTAGCGACGGCATTCCGGGCAGCGCGGGTGAGAAGGGTGAG Petição 870260016232, de 20 / 02 / 2026, pág. 60 / 126 / 55 CCGGGATTGCCGGGTCCGACTGGTCCTGCGGGCCAGAAGGGTGAA CCGGGTTCCGACGGCATCCCCGGCTCGGCGGGTGAAAAGGGCGAA CCGGGTCTGCCTGGTCCGACCGGCCCAGCGGGTCAGAAGGGTGAA CCGGGTAGCGATGGAATCCCGGGTAGCGCTGGTGAAAAGGGCGAG CCGGGCCTGCCGGGTCCGACCGGTCCGGCAGGCCAGAAGGGTGAA CCGGGTAGCGATGGTATTCCGGGTAGCGCGGGCGAAAAAGGTGAG CCGGGCTTGCCG

[00146] 11) Amino acid sequence C4P7Ec (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgsk, SEQ ID NO: 31, and the number of repeating units is 10; the amino acid sequence of C4P7Ec is SEQ ID NO: 32): Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk ID NO: 32) Base sequence C4P7Ec (SEQ ID NO: 33): GGATTTCCCGGGTCCCAGGCGCAAAAGGTGATAAA GGCAGCAAGGGCGAGGTTGGTTTCCAGGTTAGCTGGTAGCCCG GGTATCCCGGGTAGCAAGGGCTTCCCGGGTTTTCCGGGTGCTAAA GGCGACAAAGGCTCCAAGGGCGAAGTCGGTTTCCCGGGTTTGGCG GGTAGCCCGGGTATCCCGGGTAGTAAGGGCTTTCCGGGATTCCCA GGCGCGAAAGGTGACAAAGGTAGCAAGGGCGAAGTTGGCTTCCCG GGTTTGGCGGGTTCCCCGGGTATCCCGGGGTCCAAGGGCTTCCCCG Petition 870260016232, of 20 / 02 / 2026, p. 61 / 126 / 55 GATTCCCGGGCGCGAAAGGCGATAAAGGTAGCAAGGGTGAAGTG GGTTTTCCGGGTCTCGCTGGCAGCCCGGGTATTCCGGGCTCCAAGG GCTTTCCAGGCTTTCCGGGTGCGAAAGGCGATAAAGGTAGCAAGG GTGAGGTGGTTTTCCGGGTCT GCTCGAAGGGGTTCCCGGGCTTCCCGGGAGCCAAGGGTGATAAAG GTTCTAAGGGTGAGGTCGGTTTTCCGGGCCTGGCCGGTAGCCCTGG TATCCCGGGGAGCAAGGGTTTCCCGGGTTTTCCGGGTGCCAAAGG TAGCCCGGGTATTCCGGGTTCTAAGGGCTTCCCGGGTTTTCCGGGT GCGAAAGGTGACAAGGGCTCCAAGGGTGAAGTTGGTTTTCCGGGT CTGGCTGGTAGCCCGGGTATCCCGGGTAGCAAGGGCTTCCCGGT TTTCCTGGCCTGGCAGGCAGCCCGGGCATTCCGGGTTCCAAAAGGTT TTCCGGGCTTCCCGGGTGCGAAAGGTGACAAAGGCTCGAAGGGTG AGGTGGGCTTCCCGGGTCTGGCAGGTTCTCCTGGCATTCCGGGTTC GAAA

[00147] Each of the coding nucleotide sequences mentioned above was commercially synthesized. Subsequently, each of the coding nucleotide sequences above (a collagen processing enzyme cleavage site with the amino acid sequence ENLYFQ as shown by SEQ ID NO: 56 and the nucleotide sequence GAAAACCTGTATTTCCAG as shown by SEQ ID NO: 57 was added at the 5' end) was inserted between the KpnI and XhoI cleavage sites of the pET-28a-TrxHis expression vector to generate a recombinant expression plasmid.

[00148] 3. The successfully constructed expression plasmid was transformed into a competent BL21 (DE3) E. coli cell. Specifically, the process was as follows: (1) remove competent BL21 (DE3) E. coli cells from an ultra-low temperature refrigerator and place them on ice. After the Petition 870260016232, dated 20 / 02 / 2026, page 62 / 126 / 55 cells are half thawed, pipette 2 μL of the plasmid to be transformed into competent BL21 (DE3) E. coli cells and mix gently 2 to 3 times. (2) Place the mixture on ice in an ice bath for 30 minutes, then apply thermal shock in a water bath at 42°C for 45 to 90 minutes, remove the mixture and place it on ice in an ice bath for 2 minutes. (3) Transfer the mixture to a biosafety cabinet, add 700 μL of liquid LB medium and incubate at 37°C and 220 rpm for 60 min. (4) Scoop up 200 μL of the bacterial solution and spread evenly on an LB plate containing sodium ampicillin. (5) Incubate the plate in an incubator at 37°C for 15 to 17 h, until colonies of uniform size have grown.

[00149] 4. Five to six individual colonies were removed from the LB plate with transformed cells and placed in shaker flasks containing LB medium supplemented with antibiotic stock solution, followed by incubation on a shaker at 220 rpm and a constant temperature of 37°C for 7 hours. The cultured shaker flask was then cooled to 16°C, and IPTG was added to induce expression for a specific period. The bacterial solution was dispensed into centrifuge containers and centrifuged at 8,000 rpm and 4°C for 10 minutes. Bacterial cells were collected, the weight of the bacterial cells was recorded, and samples (labeled as “bacterial solution”) were collected for detection by electrophoresis.

[00150] 5. The collected bacterial cells were resuspended in an equilibrium working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole at pH 8.0), and the bacterial solution was cooled to <15°C. The solution was homogenized by two rounds of high-pressure homogenization (samples were collected after each round and labeled “homogenization 1” and “homogenization 2”, respectively), and the bacterial solution was collected after homogenization. The homogenized bacterial solution was divided into centrifuge containers and centrifuged at 17,000 rpm and 4°C for 30 minutes. The supernatant was collected, and both the Petition 870260016232, dated 20 / 02 / 2026, page 63 / 126 / 55 supernatant (labeled as “Supernatant”) when the pellets were collected for detection by electrophoresis.

[00151] 6. Purification and enzymatic digestion of recombinant humanized type IV collagen. Specifically, the processes were as follows. (1) Crude purification: a. Washing the column material (Ni6FF, Cytiva) with water for 5 CVs. b. Equilibration of the column material with equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CVs. c. Loading: loading the liquid obtained after centrifugation into the column material until the liquid flows completely through the column material and then harvesting the continuous flow (labeled “Continuous Flow”) for detection by electrophoresis. d. Washing proteins with impurities: Add 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid flows completely and collect the continuous stream of impurity wash (labeled “Impurity Wash”) for detection by electrophoresis.Collect the target protein: add 20 mL of eluate (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) and collect the continuous flow liquid (labeled: elution), detect the protein concentration and calculate the amount of protein, and perform detection by electrophoresis. f. Wash the column material with 1 M imidazole working solution (labeled “1 M Wash”). g. Wash the column material with purified water. (2) Enzymatic digestion: according to the ratio between the amount of total protein and the amount of total TEV enzyme of 50:1, add the TEV enzyme for digestion at 160°C for 4h and sample for detection by electrophoresis (labeled “After digestion”). Place the enzymatically digested protein solution in a dialysis bag and dialyze at 4°C for 2 hours, then transfer it to fresh dialysate for dialysis at 4°C overnight (labeled “Fluid Exchange”).

[00152] (3) Fine purification: a. balancing of the column material (Capto Petition 870260016232, dated 20 / 02 / 2026, p. 64 / 126 / 55 a. Equilibration: Equilibration of the column material using solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) at a flow rate of 10 ml / min. b. Loading: Loading the sample at a flow rate of 5 ml / min and collecting the continuous flow (labeled “QFL”) and performing detection by electrophoresis. c. Gradient elution: Setting up 0 to 15% of solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 min followed by retention for 3 CVs, 15 to 30% of solution B for 2 min followed by retention for 3 CVs, 30 to 50% of solution B for 2 min followed by retention for 3 CVs, respectively, collecting the peak and performing detection by electrophoresis (labeled “Washing with solution B”). d. Washing the column material. The protein was stored at 4°C.

[00153] 7. Electrophoretic detection

[00154] Specifically, the processes were as follows. 40 μL of sample solution were collected and 10 μL of 5x protein loading buffer (250 mM Tris-HCl at pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) were added. The mixture was heated in boiling water at 100°C for 10 minutes, and then 10 μL were loaded into each well of an SDS-PAGE protein gel. The gel was run at 80 V for 2 hours, stained for 20 minutes using Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) and then decolorized using protein decolorizing solution (10% acetic acid, 5% ethanol).

[00155] Figure 1 shows the electrophoresis detection results for C4P7Ca. Figure 2 shows the electrophoresis detection results for C4P7Cb. Figure 3 shows the electrophoresis detection results for C4P7Cc. Figure 4 shows the results of detection by electrophoresis. C4P7Cd. Figure 5 shows the results of detection by electrophoresis. C4P7Ce. Figure 6 shows the results of detection by electrophoresis. Petition 870260016232, dated 20 / 02 / 2026, page 65 / 126 / 55 Figure 7 shows the electrophoresis detection results for C4P7Cg. Figure 8 shows the electrophoresis detection results for C4P7Ch. Figure 9 shows the electrophoresis detection results for C4P7Ea. Figure 10 shows the electrophoresis detection results for C4P7Eb. Figure 11 shows the electrophoresis detection results for 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, demonstrating that the proteins are correctly expressed. Example 2: Mass spectrometry detection of recombinant humanized type IV collagens Table 1: Experimental method Instrument Name Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF / TOF) mass spectrometer Ultraflextreme™, Brucker, Germany Matrix CHCA Laser Energy 125 Data Retrieval Software Mascot Retrieval Species All species Retrieval Database Sequences provided as library

[00156] Protein samples (C4P7Cf and C4P7Ch collagens) were reduced with DTT and alkylated with iodoacetamide, followed by the addition of trypsin for overnight enzymatic digestion. The peptide fragments obtained after enzymatic digestion were desalted using C18ZipTip, mixed with the α-cyano-4-hydroxycinnamic acid (CHCA) matrix, and placed on a plate. Finally, matrix-assisted laser desorption / time-of-flight ionization mass spectrometry (MALDI-TOF / TOF Ulraflextreme™, Bruker, Germany) was used for analysis (see Protein J. 2016;35:212-7 for peptide fingerprinting technology).

[00157] Data retrieval was performed using the MS / MS Ion Search page of the local Mascot website. Protein identification results were obtained based on primary mass spectrometry of Petition 870260016232, dated 20 / 02 / 2026, page 66 / 126 / 55 peptide fragments generated after enzymatic digestion. Detection parameters: enzymatic digestion with trypsin, allowing two cleavage sites to be missed. Cysteine ​​alkylation was defined as a fixed modification, and methionine oxidation was defined as a variable modification. The database used for identification was NCBprot. Table 2: Molecular weights and corresponding polypeptides detected by mass spectrometry of recombinant humanized type IV collagen C4P7Cf Start-End Observed value Mr (expected value) Peptide 241 to 276 3353.6817 3352.6744 GSKGEVGFPGLAGSPGIPGSKGEQGFMGPP GPQGAK (SEQ ID NO: 39) 244 to 276 3065.6004 3064.5932 GEVGFPGLAGSPGIPGSKGEQGFMGPPGPQ GAK (SEQ ID NO: 40) 244 a 279 3381.6741 3380.6668 GEVGFPGLAGSPGIPGSKGEQGFMGPPGPQ GARGDK (SEQ ID NO: 41) 262 a 276 1457.6553 1456.6480 GEQGFMGPPGPQGAK (SEQ ID NO: 42) 262 a 276 1473.6731 1472.6658 GEQGFMGPPGPQGAK (SEQ ID NO: 42) 262 a 279 1773.7801 1772.7728 GEQGFMGPPGPQGAKGDK (SEQ ID NO: 43) 277 a 300 2199.0695 2198.0622 GDKGSKGEVGFPGLAGSPGIPGSK (SEQ ID NO: 44) 280 a 300 1898.9500 1897.9427 GSKGEVGFPGLAGSPGIPGSK (SEQ ID NO: 45) 280 a 312 3061.5039 3080.4966 GSKGEVGFPGLAGSPGIPGSKGEQGFMGPP GPQ (SEQ ID NO: 46) 280 a 312 3097.4825 3096.4752 GSKGEVGFPGLAGSPGIPGSKGEQGFMGPP GPQ (SEQ ID NO: 46) 283 a 300 1626.8268 1625.8196 GEVGFPGLAGSPGIPGSK (SEQ ID NO: 47) 283 a 312 2825.2583 2824.2510 GEVGFPGLAGSPGIPGSKGEQGFMGPPGPQ (SEQ ID NO: 48); 301 a 312 1217.5133 1216.5060 GEQGFMGPPGPQ (SEQ ID NO: 49)

[00158] The coverage rate of the detected polypeptide segments was 99.04% compared to the theoretical sequence (GAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKG SKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFP GLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIP GSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGF Petition 870260016232, dated 20 / 02 / 2026, page 67 / 126 / 55 MGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQG AKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGS KGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQ, SEQ ID NO: 5, where the covered part is underlined), therefore, the detection results were very reliable. Table 3: Molecular weights and corresponding polypeptides detected by mass spectrometry of recombinant humanized type IV collagen C4P7Ch Start-End Observed Value Mr (Expected Value) Peptide 247 to 273 2469.2114 2468.2041 GSKGEVGFPGLAGSPGIPGSKGEQGAK (SEQ ID NO: 50) 250 to 273 2197.0655 2196.0782 GEVGFPGLAGSPGIPGSKGEQGAK (SEQ ID NO: 51) 250 to 276 2497.1996 24961923 GEVGFPGLAGSPGIPGSKGEQGAKGDK (SEQ ID NO: 52) 277 to 297 1898.9493 1897.9421 GSKGEVGFPGLAGSPGIPGSK (SEQ ID NO: 53) 280 at 297 1626.8349 1625.8276 GEVGFPGLAGSPGIPGSK (SEQ ID NO: 54) 280 to 300 1940.9073 1939.9000 GEVGFPGLAGSPGIPGSKGEQ (SEQ ID NO: 55)

[00159] The coverage rate of the detected polypeptide segments was 98% compared to the theoretical sequence (GAKGDKGSKGEVGFPGLAGSPGIGGSKGEQGAKGDKGSKGEVGFPG LAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAK GDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGS PGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKG SKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPG SKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGE VGFPGLAGSPGIPGSKGEQ, SEQ ID NO: 2, where the covered part is underlined), therefore, the detection results were very reliable. Example 3: Detection of Bioactivity of Recombinant Humanized Type IV Collagen

[00160] Methods for detecting collagen activity can be found in 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 Petition 870260016232, dated 20 / 02 / 2026, p. 68 / 126 / 55 Biochem. 136, 643-649(2004). The specific methods were performed as follows: (1) The ultraviolet absorption method was used to detect the concentration of protein samples to be detected, including bovine type I collagen (National Institutes for Food and Drug Control, No. 380002), recombinant humanized collagens C4P7Cf and C4P7Ch provided by the present description.

[00161] Specifically, the ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and protein concentrations were calculated using the empirical formula C (lg / mL) = 144 x (A215A225). Note that detection should be performed when A215 < 1.5. The principle of this method is that the characteristic absorption of peptide bonds under far-ultraviolet light is detected, which is not affected by chromophore content, with few interfering substances. The method is easy to operate, so it is suitable for the detection of human collagen and its analogues that do not develop color with Coomassie brilliant blue. (See reference Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After detecting the protein concentration, the concentration of all proteins to be tested was adjusted to 0.5 mg / mL with PBS.

[00162] (2) Sample preparation: the stock sample solution was used directly to conduct the experiment. The positive control of bovine type I collagen (PC) was diluted to 1 mg / ml with D-PBS for future use; and the negative control was D-PBS buffer (NC).

[00163] (3) Coating: different concentrations of collagen (C4P7Cf or C4P7Ch) and the positive control and negative control were added to an ELISA plate in a volume of 100 μL per well, and 5 replicated wells were placed in each group and then incubated at 40C overnight. Petition 870260016232, dated 20 / 02 / 2026, page 69 / 126 54 / 55

[00164] (4) Blocking: The supernatant was discarded and 100 pL of 1% BSA (heat inactivated at 56°C for 30 minutes) was added, followed by incubation at 37°C for 60 minutes. The supernatant was discarded and the plate was washed 3 times with D-PBS solution.

[00165] (5) Cell seeding: 105 well-cultured 3T3 / NIH cells resuspended in D-PBS were added to each well and incubated at 37°C for 120 minutes. Each well was washed 3 times with D-PBS solution.

[00166] (6) Detection: absorbance at OD450 nm was detected using the CCK8 detection kit (manufacturer Beyotime Biotech Inc., product catalog number C0038). The degree of cell adhesion was calculated according to the following formula. The rate of cell adhesion can reflect the cell adhesion capacity of collagen. The greater the cell adhesion capacity, the better the external environment can be provided to the cells in a short time to facilitate cell adhesion. OD, -ODnp —-------OD2-OD0em where: P: relative cell adhesion ratio; ODi: the average ultraviolet absorbance at 450 nm of all replicated wells of the tested collagen sample; OD2: Average ultraviolet absorbance at 450 nm for all replicated wells of the control collagen sample; OD0: Average ultraviolet absorbance at 450 nm for all replicated wells from the blank control group.

[00167] (7) Statistical analysis: The statistical difference between the target recombinant humanized collagen and the negative control was statistically analyzed using the two-tailed t-test, where *, P < 0.05; * *, P < 0.01; * * *, P < 0.001.

[00168] The results were shown in Figures 11 and 12. In Petition 870260016232, dated 20 / 02 / 2026, page 70 / 126 / 55 comparison with the D-PBS group, the positive control had a significant effect on promoting cell adhesion, and recombinant humanized collagen C4P7Cf and C4P7Ch also promote cell adhesion.

[00169] Although the present description has been described with reference to illustrative embodiments, those skilled in the art will recognize that various other alterations, omissions, and / or additions may be made and that elements of the embodiments described may be substituted for substantial equivalents without departing from the spirit and scope of the description. Furthermore, many modifications may be made to adapt specific situations or materials to the precepts of this description without departing from the scope of this description. Consequently, it is not intended that the description be limited to the specific embodiments described herein for the purpose of carrying out the description, but rather that the description include all embodiments that fall within the scope of the appended claims. Petition 870260016232, dated 20 / 02 / 2026, p. 71 / 126

Claims

1 / 2 CLAIMS 1. Recombinant collagen, characterized in that the amino acid sequence of the recombinant collagen is SEQ ID NO:

2.

2. Nucleic acid, characterized in that it encodes recombinant collagen as defined in claim 1, wherein the nucleotide sequence of the nucleic acid is SEQ ID NO:

3.

3. Vector, characterized in that it comprises the nucleic acid as defined in claim 2.

4. Host cell, characterized in that it comprises the nucleic acid as defined in claim 2, or the vector as defined in claim 3, wherein the host cell is selected from the group consisting of yeast cells or Escherichia coli cells.

5. Composition, characterized in that it comprises recombinant collagen as defined in claim 1. 6.Composition according to claim 5, characterized in that the composition is one or more of a biological dressing, a human bionic material, a material for plastic or aesthetic surgery, an organoid culture material, a cardiovascular stent material, a coating material, a filler material for tissue injection, an ophthalmic material, a biomaterial for obstetrics and gynecology, a material for nerve repair and regeneration, a material for liver tissue and a material for blood vessel repair and regeneration, a biomaterial for 3D-printed artificial organs, a cosmetic raw material, a pharmaceutical excipient and a food additive.

7. Composition according to claim 6, characterized in that the composition is a composition for topical, injectable or oral use. 8.Composition according to claim 6, characterized in that the composition is a composition in the form of a solution, Petition 870260065553, dated 03 / 07 / 2026, page 11 / 15 2 / 2 a lyophilized powder, a gel, a sponge or a fiber. 9.Use of recombinant collagen as defined in claim 1, nucleic acid as defined in claim 2, vector as defined in claim 3, host cell as defined in claim 4 and / or composition as defined in any of claims 5 to 8, characterized by being used in the manufacture of one or more of the following: biological dressing, human bionic material, material for plastic or cosmetic surgery, organoid culture material, cardiovascular stent material, coating material, filler material for tissue injection, ophthalmic material, biomaterial for obstetrics and gynecology, nerve repair and regeneration material, liver tissue material and blood vessel repair and regeneration material, biomaterial for 3D printed artificial organs, cosmetic raw material, pharmaceutical excipient and food additive. 10.In vitro method for facilitating cell adhesion for non-therapeutic purposes, characterized in that it comprises the step of contacting recombinant collagen, as defined in claim 1, with an in vitro cell.

11. Method according to claim 10, characterized in that the cell is an animal cell.

12. Method for producing recombinant collagen as defined in claim 1, characterized in that it comprises: (1) incubating the host cell, as defined in claim 4, under suitable culture conditions; (2) collecting the host cell and / or the culture medium comprising recombinant collagen; and (3) purification of the recombinant collagen.

13. Method according to claim 12, characterized in that the host cell is an E. coli cell. Petition 870260065553, dated 03 / 07 / 2026, p. 12 / 15

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