Method for biosynthesis of human structural material type xvii collagen

Abstract

Provided is a method for biosynthesis of a human structural material type XVII collagen. The collagen comprises the amino acid sequence shown in SEQ ID NO: 2 or a variant amino acid sequence after mutation of the amino acid sequence. The variant amino acid sequence retains the function of the amino acid sequence as shown in SEQ ID NO: 2. The collagen can promote cell adhesion and has a triple helix structure.

Description

Method for biosynthesizing type XVII collagen, a structural material for the human body

[0001] This application claims the priority benefit of a Chinese invention patent application filed on January 8, 2024, with application number 202410026356.6 and titled “Method for biosynthesizing type XVII collagen as a structural material for the human body”. Technical Field

[0002] The present application relates to the field of proteins or polypeptides, and in particular to collagen, its preparation method and use. Background Art

[0003] Collagen is a protein widely distributed throughout human connective tissue and is the most abundant protein in the human body, comprising 25% to 35% of the total protein content. Its primary functions include maintaining the extracellular environment, upholding the normal physiological functions of tissues and organs, and repairing body damage. Collagen is a natural biological resource, possessing unmatched biocompatibility, cell-supporting elasticity, and biodegradability. Therefore, collagen is widely used in industries such as medicine and cosmetics.

[0004] Natural collagen molecules form a unique superhelical structure, a left-handed helix based on three repeating amino acid residues, typically arranged in the form of Gly-X-Y. Gly is essential for the formation of hydrogen bonds within collagen, and its lack of side chains allows for dense collagen packing, maintaining skin tension and elasticity.

[0005] With the widespread application of genetic engineering techniques in recent years, researchers have created various types of recombinant collagen. For example, recombinant collagen can be constructed by selecting short amino acid sequences derived from natural human collagen. These recombinant collagens have advantages such as low immunogenicity, high biological activity, and excellent stability. In theory, the shorter the amino acid sequence of such recombinant collagen, the better its transdermal absorption performance. However, shorter amino acid sequences are not necessarily better. There is no theoretical guidance in the prior art on how to design short amino acid sequences that can enhance transdermal absorption of the recombinant collagen.

[0006] Type XVII collagen (COL17) is a transmembrane protein primarily expressed in basal keratinocytes of the epidermis. COL17 expression on hemidesmosomes in the epidermal basal membrane zone is required for epidermal-dermal adhesion, as congenital COL17 deficiency leads to junctional epidermolysis bullosa. In addition to promoting epidermal-dermal adhesion, COL17 serves as a niche for hair follicle stem cells, regulates proliferation of the interfollicular epidermis, and is present along the non-hemidesmosomal plasma membrane of epidermal basal keratinocytes. COL17's role in stem cell maintenance and its involvement in signaling pathways contribute to the maintenance of stable adhesion between the dermis and epidermis. The function and stability of collagen depend on the formation of triple helices of distinct polypeptide chains. Triple helical structure formation is believed to be dependent on specific triple helical regions. However, the physiological relevance of these coiled-coil structures is poorly understood. COL17 and other members of the membrane-associated collagen subfamily undergo triple helical assembly from the N-terminus (membrane-proximal) to the C-terminus.

[0007] There is a need in the art for new type XVII collagen and methods for their preparation.

[0008] Summary of the Invention

[0009] To address current needs, the inventors have provided novel type XVII collagen. The type XVII collagen described herein has the ability to promote cell adhesion and possesses a triple helical structure. The present invention also provides a method for biosynthesizing type XVII collagen, a structural material for the human body.

[0010] In a first aspect, a collagen is provided, which comprises a plurality of repeating units, wherein the repeating units comprise the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence shown in SEQ ID NO: 1; the number of repeating units is 10-20, 12-18 or 13-16.

[0011] In one embodiment, the number of repeating units is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0012] In one embodiment, each repeat unit is directly linked or connected through a linker of one amino acid or multiple amino acid residues.In one embodiment, the linker comprises 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0013] In one embodiment, the mutation is a substitution, insertion, deletion or addition. In one embodiment, the substitution is a conservative amino acid substitution.

[0014] In one embodiment, the collagen is of human origin. In one embodiment, the collagen has a triple helical structure. In one embodiment, the collagen has cell adhesion properties. In one embodiment, the collagen is recombinant collagen, recombinant humanized collagen, or recombinant humanized type XVII collagen.

[0015] In one embodiment, the collagen comprises the following amino acid sequence:

[0016] (1) the amino acid sequence shown in SEQ ID NO: 2;

[0017] (2) an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; or

[0018] (3) An amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence shown in SEQ ID NO: 2.

[0019] In one embodiment, the mutation is a substitution, insertion, deletion or addition. In one embodiment, the substitution is a conservative amino acid substitution.

[0020] In a second aspect, a nucleic acid is provided that encodes the collagen described herein.

[0021] In one embodiment, the nucleic acid has the nucleotide sequence shown in SEQ ID NO:3.

[0022] In a third aspect, a vector is provided, comprising a nucleic acid according to the present invention. In one embodiment, the vector comprises nucleotides encoding a purification tag, nucleotides encoding a leader and / or a regulatory element.

[0023] In one embodiment, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag.

[0024] In one embodiment, the regulatory element is selected from a promoter, a terminator and / or an enhancer.

[0025] In a fourth aspect, a host cell is provided, comprising a nucleic acid as described herein or a vector as described herein.

[0026] 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 / or the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.

[0027] In a fifth aspect, a method for producing collagen is provided, comprising:

[0028] (1) culturing the host cell described herein under appropriate culture conditions;

[0029] (2) harvesting host cells and / or culture medium containing collagen; and

[0030] (3) Purification of collagen, for example, including (1) crude purification of collagen on a Ni affinity chromatography column; (2) enzymatic cleavage with the addition of collagen tools; and / or (3) fine purification of collagen using an ion exchange column.

[0031] In a sixth aspect, a composition is provided, comprising the collagen, nucleic acid, vector and / or host cell described herein.

[0032] In one embodiment, the composition is a pharmaceutical composition or a cosmetic composition.

[0033] In one embodiment, the composition is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0034] In one embodiment, the composition comprises a pharmaceutically and / or cosmetically acceptable carrier.

[0035] In one embodiment, the composition is a solid, liquid or gel composition.

[0036] In one embodiment, the composition is an oral and / or topically administrable composition, preferably a topical composition.

[0037] In one embodiment, the composition is a kit.

[0038] In one embodiment, the composition is a liquid formulation comprising the collagen described herein and a pharmaceutically and / or cosmetically acceptable carrier.

[0039] In one embodiment, the carrier is a buffer, such as D-PBS buffer or PBS buffer.

[0040] In a seventh aspect, a method for promoting cell adhesion or attachment is provided, the method comprising contacting cells with the collagen and / or composition herein.

[0041] In an eighth aspect, provided are uses of the collagen, nucleic acid, vector, host cell and / or composition described herein in biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials, vascular repair and regeneration materials, or 3D printed artificial organ biomaterials.

[0042] In a ninth aspect, provided is the use of the collagen and / or composition described herein in the preparation of a drug or kit for promoting cell adhesion or cell attachment.

[0043] The advantages of the present invention include: the collagen described herein is a new humanized type XVII collagen, which has the effect of promoting cell adhesion and has a triple helical structure; the recombinant humanized collagen C17T15 herein has higher (more than 2 times) cell adhesion activity than bovine type I collagen, achieving unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 shows the electrophoresis pattern of recombinant humanized type XVII collagen C17T15.

[0045] FIG2 shows the mass spectrum of recombinant humanized type XVII collagen C17T15.

[0046] FIG3 shows cell adhesion of recombinant humanized type XVII collagen C17T15.

[0047] FIG4 shows the circular dichroism spectrum of recombinant humanized type XVII collagen C17T15.

[0048] FIG5 shows the cell viability of recombinant humanized type XVII collagen C17T15 in Hela cells. DETAILED DESCRIPTION

[0049] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0050] As used herein, recombinant humanized collagen is a full-length or partial amino acid sequence fragment encoded by a sex-specific human collagen gene, or a combination of functional human collagen fragments, prepared by DNA recombinant technology. In this article, recombinant humanized collagen is recombinant humanized type XVII collagen, which is a peptide or polypeptide composed of multiple amino acid residues linked by peptide bonds.

[0051] As used herein, "one or more" can be any suitable integer. In the case of collagen mutations (e.g., substitutions, deletions, insertions, or additions), "one or more" is a number readily determined by those skilled in the art, such as 1-90 and any integers and ranges therebetween, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, etc.

[0052] As used herein, "nucleic acid" refers to a plurality of nucleotides linked by internucleotides. The internucleotide linkages may be, for example, phosphodiester bonds. The nucleic acid herein may comprise a polynucleotide encoding a polypeptide of the present invention. To facilitate subsequent processing of the polypeptide, the nucleic acid of the present invention may further comprise nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag, and, when necessary, a nucleotide sequence encoding a leader sequence.

[0053] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. A vector can contain a nucleic acid of the present invention to facilitate introduction into cells for expression. A vector can contain expression control elements, such as a promoter, terminator, and / or enhancer, operably linked to the nucleic acid.

[0054] As used herein, the term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell can be a eukaryotic cell or a prokaryotic cell. For example, a eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell can be an Escherichia coli cell.

[0055] As used herein, "biological dressing" is a new type of medical dressing used for wound repair and treatment. It is a specialized medical material made by professional craftsmen from biomaterials and combined with drugs or other therapeutic substances. The collagen in the biological dressing forms a protective layer on the wound surface, promoting cell proliferation and regeneration, and accelerating wound healing.

[0056] As used herein, "bionic materials" refer to materials developed and manufactured to mimic the various characteristics or properties of living organisms. Artificial materials designed and manufactured to mimic the operating modes of living systems and the structural laws of biological materials are generally referred to as bionic materials. "Human bionic materials" refer to materials developed and manufactured to mimic the various characteristics or properties of the human body. Artificial materials designed and manufactured to mimic the operating modes of living systems and the structural laws of biological materials are generally referred to as bionic materials.

[0057] As used herein, "organoid culture material" refers to artificial materials used to culture and construct organoid functions to address the needs of organ transplantation and replacement.

[0058] As used herein, "biomaterial" refers to materials that are compatible with living tissue and are commonly used to create artificial organs or tissue replacements. "3D-printed artificial organ biomaterial" refers to the biomaterial used in 3D-printed artificial organs.

[0059] As used herein, the degree of association between two amino acid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use as in EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, people such as Rice, 2000, Trends Genet. [genetics trend] 16:276-277) (preferred 5.0.0 version or more recent version) Niederman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using non-simplified option to obtain) of Nieder, who is labeled as " longest identity ", is used as identity percentage and is calculated as follows:

[0060] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment)

[0061] For purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented by the Needleman program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needleman labeled "longest identity" (obtained using the non-simplified option) is used as percent identity and is calculated as follows:

[0062] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0063] In the context of the present invention, conservative amino acid substitutions or conservative substitutions can be defined by substitutions within the amino acid classes reflected in one or more of the following tables:

[0064] Conserved amino acid residues:

[0065] Physical and functional classification of alternative amino acid residues:

[0066] Recombinant humanized type XVII collagen

[0067] In this article, the recombinant humanized type XVII collagen of the present invention may have certain mutations. For example, the amino acid sequence of one or more of these parts may have substitutions, deletions, additions or insertions of amino acid residues. That is, the present invention can use variants as long as the variants retain the activity of promoting cell adhesion and / or proliferation. Specifically, the variants can have a certain percentage identity with the specified sequence, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity. The specified sequence can be any sequence of the present invention, such as SEQ ID NO: 1 or 2, but it is preferred that these variants retain the function of the recombinant humanized type XVII collagen of the present invention. The recombinant humanized type XVII collagen of the present invention can have good cell adhesion efficacy and have a triple helical structure.

[0068] The recombinant humanized type XVII collagen of the present invention can be prepared by any suitable means, for example, by synthesis. Preferably, the recombinant humanized type XVII collagen of the present invention can be prepared by recombinant means.

[0069] The collagen of the present invention may have a triple helical region, that is, a triple helical collagen structure consisting of three identical collagen chains. For example, collagen may have a flexible triple helical region. The collagen of the present invention can form a triple helical structure. Determination has shown that the recombinant humanized type XVII collagen C17T15 of the present invention can form a triple helical structure. The triple helical structure of collagen is formed by three intertwined polypeptide chains. Each polypeptide chain is linked by many amino acids, with amino acids such as glycine, proline, and hydroxyproline being particularly important in the structure of collagen. Each of these amino acids can form hydrogen bonds, which link the three polypeptide chains together to form a triple helical structure. In this process, each polypeptide chain intertwines with the other two in the same manner. Research has shown that the triple helical structure of collagen plays a crucial role in its thermal stability. The three-dimensional structure formed by the intertwining of polypeptide chains through hydrogen bonds is extremely stable. Extensive experimental results have shown that very high temperatures or pH levels are required to disrupt the triple helical structure of collagen. In vivo, the thermal stability of collagen provides many benefits. For example, due to its high thermal stability, collagen can maintain its stability and function in the human body for a long time. This stability also provides collagen with the endurance to withstand the stress generated by daily physical activity and metabolic processes without breaking down or decomposing.

[0070] Composition

[0071] The recombinant humanized type XVII collagen of the present invention can be prepared as a composition. The composition may comprise the recombinant humanized type XVII collagen described herein, a nucleic acid, a vector and / or a host cell. The composition may also comprise a pharmaceutically and / or cosmetically acceptable carrier or solvent. The composition may be a pharmaceutical composition or a cosmetic composition, used for pharmaceutical and / or cosmetic purposes. For example, the composition is one or more of a biological dressing, a human biomimetic material, a plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient and a food additive.

[0072] The cosmetic composition may be applied to any part of the body without particular limitation, and may be applied to the face, hands, legs, trunk, etc. The form of the composition is not particularly limited, as long as it can achieve the intended function. For example, the composition may be a solid, liquid, or gel composition.

[0073] The composition can be used in any suitable manner, for example, for oral and / or topical administration. The composition can also be prepared as a test kit. The test kit can include other ingredients, for example, auxiliary ingredients, such as buffers, and include instructions for use. In particular, the composition can be formulated into a suitable formulation, such as a liquid formulation. The formulation can include a buffer, for example, D-PBS buffer or PBS buffer.

[0074] Methods and uses

[0075] Provided herein is a method for causing cells to adhere or adhere, the method comprising contacting cells with recombinant humanized type XVII collagen, compositions and / or liquid formulations as described herein. The method of the present invention can be performed in vitro to increase cell adhesion to a culture vessel. Alternatively, the method of the present invention can also be performed in vivo. The present invention also provides the use of the recombinant humanized type XVII collagen, compositions and / or liquid formulations described herein in the preparation of a medicament and / or a kit for increasing cell adhesion. In addition to the collagen described herein, the kit may also include a suitable carrier, diluent or excipient, etc., as well as instructions for using the collagen.

[0076] Example

[0077] The following examples are provided to illustrate the present invention. It should be understood by those skilled in the art that the examples are merely illustrative and non-limiting. The present invention is limited only by the scope of the appended claims.

[0078] Example 1: Construction, expression and screening of humanized type XVII collagen fragments

[0079] 1. Perform large-scale functional region screening to obtain the following target gene functional regions of humanized type XVII collagen.

[0080] C17T15 amino acid sequence: ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq ghkgekgdkgdq (SEQ ID NO: 2; the repeating unit is ghkgekgdkgdq, SEQ ID NO: 1).

[0081] The inventors designed the C17T15 nucleotide sequence based on the C17T15 amino acid sequence.

[0082] C17T15 nucleotide sequence:

[0083] 2. Construction of genetically engineered Escherichia coli

[0084] The C17T15 nucleotide sequence is cloned into an expression vector, and then the expression vector is transformed into an Escherichia coli expression strain, and the genetically engineered Escherichia coli is obtained by screening.

[0085] Specifically, according to the amino acid sequence of C17T15 (SEQ ID NO: 2), the codon gene SEQ ID NO: 3 of its Escherichia coli preference was optimized and selected. The nucleotide sequence of SEQ ID NO: 3 was synthesized. The C17T15 gene fragment was inserted into the pET-32a expression vector (Beijing Liuhe Huada Gene Technology Co., Ltd.) through the restriction sites of Kpn I (NEB Company, Article No.: R0136L) and Xho I (NEB Company, Article No.: R0146L), and the pET-32a-C17T15 expression vector was constructed. The expression vector was introduced into Escherichia coli BL21 (DE3), and positive Escherichia coli genetically engineered bacteria were screened. The above operations were entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd.

[0086] 3. Fermentation culture of genetically engineered Escherichia coli

[0087] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3). The specific process is as follows:

[0088] (1) Take out the competent E. coli cells BL21 (DE3) from the ultra-low temperature refrigerator and place them on ice. Take 2 μl of the plasmid to be transformed and add it to the competent cells BL21 (DE3). Mix gently 2-3 times.

[0089] (2) Place the mixture on ice for 30 minutes, then heat shock in a 42°C water bath for 45-90 seconds, remove it and place it on ice for 2 minutes.

[0090] (3) Transfer the tube to a biosafety cabinet and add 700 μl of liquid LB medium. Then, incubate the tube at 37°C and 220 rpm for 60 min.

[0091] (4) Take 200 μl of bacterial solution and spread it evenly on LB plate containing ampicillin sodium.

[0092] (5) Incubate the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0093] (6) Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing LB medium containing antibiotic stock solution. Incubate in a constant temperature shaker at 220 rpm and 37°C for 7 hours. Then cool the shake flask to 16°C. After adding IPTG to induce expression for a period of time, the bacterial solution is divided into centrifuge bottles and centrifuged at 8000 rpm and 4°C for 10 minutes. Collect the bacteria, record the bacterial weight, and take samples for electrophoresis.

[0094] (7) Resuspend the collected bacteria in a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), cool the bacterial solution to ≤15°C, homogenize it, and homogenize it twice under high pressure. After completion, collect the bacterial solution. The homogenized bacterial solution is divided into centrifuge bottles and centrifuged at 17,000 rpm and 4°C for 30 minutes. The supernatant is collected and the supernatant and precipitate are taken for electrophoresis detection.

[0095] (8) Purify and digest C17T15. The specific process is as follows: (1) Crude purification: a. Wash the column (Ni6FF, Cytiva) with water for 5 CV. b. Equilibrate the column with equilibration solution (200mM sodium chloride, 25mM Tris, 20mM imidazole, pH8.0) for 5 CV. c. Load the sample: Add the supernatant after centrifugation to the column until the liquid flows out, and take the flow-through for electrophoresis. d. Clean the impurities: Add 25mL of washing solution (200mM sodium chloride, 25mM Tris, 20mM imidazole) until the liquid flows out, and take the wash flow-through for electrophoresis. e. Collect the target protein: Add 20mL of eluent (200mM sodium chloride, 25mM Tris, 250mM imidazole, pH8.0), collect the flow-through, detect the protein concentration, calculate the protein amount, and perform electrophoresis. f. Clean the column with 1M imidazole working solution. g. Wash the column with purified water. (2) Enzymatic digestion: Add TEV enzyme at a ratio of 50:1 between the total amount of protein and the total amount of TEV enzyme, digest at 16°C for 4 hours, and take samples for electrophoresis detection. Place the protein solution after enzymatic digestion in a dialysis bag, dialyze at 4°C for 2 hours, and then transfer to new dialysate for overnight dialysis at 4°C. (3) Purification: a. Equilibrate the column (Ni6FF, Cytiva): Equilibrate the column with solution A (20mM Tris, 20mM sodium chloride, pH8.0) at a flow rate of 10ml / min. b. Loading: Load the sample at a flow rate of 5ml / min, collect the flow-through, and perform electrophoresis detection. c. Gradient elution: Set the gradient to 0-15% Solution B (20 mM Tris, 1 M NaCl, pH 8.0) for 2 min and hold for 3 CVs, 15-30% Solution B for 2 min and hold for 3 CVs, 30-50% Solution B for 2 min and hold for 3 CVs, and 50-100% Solution B for 2 min and hold for 3 CVs. Collect peaks and perform electrophoresis for purified protein. d. Wash the column. Store the protein at 4°C.

[0096] Concentration determination: Accurately measure an appropriate amount of sample, dilute 10-50 times with eluent, and stir thoroughly with a glass rod. Measure the absorbance at 280 nm using a UV-visible spectrophotometer. Calculate the protein concentration using the formula C (mg / ml) = A280 × absorbance coefficient × dilution factor (Note: The absorbance value must be between 0.1 and 1).

[0097] The specific process of electrophoresis detection is as follows: take 40μl of sample solution, add 10μl 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in boiling water at 100℃ for 10 minutes, then add 10μl per well to SDS-PAGE protein gel, run at 80V for 2 hours, use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20 minutes, and then use protein destaining solution (10% acetic acid, 5% ethanol) for decolorization. The experimental results are shown in Figure 1. The left lane is the target protein obtained after enzyme digestion, the second left lane is the remaining carrier protein after enzyme digestion, and the rightmost lane is the marker. As shown in the figure, the target protein band is single and free of contaminant protein bands.

[0098] The specific process of mass spectrometry detection is as follows:

[0099] A. The purified protein is dialyzed against ultrapure water to remove salt components and then freeze-dried in a vacuum to form protein powder.

[0100] B. Dissolve the protein powder and dilute to 0.1-10 pmol / μl in ultrapure water or matrix buffer containing 50% acetonitrile and 0.1% trifluoroacetic acid.

[0101] C. Mix the protein sample solution and saturated matrix solution in a 1:1 ratio.

[0102] D. Take 1 μl of the above mixture and add it to the sample target, and air dry it.

[0103] E. Place the sample target containing the protein standard and sample protein into a MALDI-TOF-MS mass spectrometer using ESI as the ion source. Detection mode: Positive ion. Precursor ion scan range: 500-4000 m / z. Compare the measured molecular weight of the target protein with the relative molecular weight deduced from the protein's amino acid sequence.

[0104] The mass spectrometry results of the purified recombinant humanized collagen C17T15 are shown in Figure 2. As shown in Figure 2, the theoretical molecular weight of the collagen C17T15 of the present invention is 18577.43, and the highest peak on the mass spectrum shows an actual molecular weight of 18577.1 kDa, which is consistent with the theoretical molecular weight.

[0105] Example 2: Bioactivity Detection of Recombinant Humanized Collagen

[0106] The adhesion activity assay for recombinant humanized collagen can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0107] (1) The concentration of the protein samples to be tested was detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Institute, No.: 380002) and the recombinant humanized collagen C17T15 provided by the present invention. Specifically, the ultraviolet absorption of the samples at 215nm and 225nm was measured respectively, and the protein concentration was calculated using the empirical formula C (μg / mL) = 144×(A215-A225). Note that the test should be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far ultraviolet light, it is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After the protein concentration was detected, the concentration of all the proteins to be tested was adjusted to 0.5 mg / mL with PBS.

[0108] (2) Add 100 μL of various protein solutions (bovine type I collagen or recombinant humanized collagen C17T15) or D-PBS solution (blank control group) to a 96-well plate.

[0109] (3) Add 10 5 3T3 cells in good culture condition were incubated at 37°C for 60 min.

[0110] (4) Wash each well 4 times with PBS.

[0111] (5) Use LDH detection kit (Roche, 04744926001) to detect the absorbance at OD492nm. Based on the value of blank control, the cell adhesion rate can be calculated. The calculation formula is as follows

[0112] Where:

[0113] P: relative cell adhesion ratio;

[0114] OD1: average absorbance value of each replicate well of the test collagen sample at OD492nm;

[0115] OD2: average absorbance of each replicate well of the control collagen sample at OD492nm;

[0116] OD0: The average absorbance of each replicate well in the blank control group at OD492nm.

[0117] The cell adhesion rate reflects the adhesion activity of each protein. The higher the activity of the protein, the better the external environment it can provide to cells in a short time, helping them to adhere to the wall.

[0118] The results are shown in Figure 3. The comparison shows that the recombinant collagen C17T15 of the present invention exhibits superior bioadhesion activity compared to bovine type I collagen (PC group, 0.5 mg / ml). Figure 3 depicts the relative cell adhesion activity of recombinant humanized collagen C17T15 relative to bovine type I collagen, demonstrating that recombinant humanized collagen C17T15 exhibits higher (more than two-fold) cell adhesion activity than bovine type I collagen. The high cell adhesion activity of recombinant humanized collagen C17T15 is unexpected.

[0119] Example 3: Circular dichroism of recombinant humanized collagen C17T15

[0120] 1) Sample preparation

[0121] Prepare 1X phosphate buffer solution (PBS) by dissolving 8 g NaCl, 0.2 g KCl, 3.62 g Na2HPO4.12H2O, and 0.24 g KH2PO4 in 800 ml distilled water, adjusting the pH of the solution to 7.4 with HCl, and adding water to 1 L. After autoclaving, store at room temperature. If stored for more than 1 week, filter through a 0.45 filter before use.

[0122] Sample dissolution: Dissolve the freeze-dried flocculent sample in PBS - each bottle contains 4 mg of recombinant humanized collagen C17T15 protein, draw 2 mL of PBS into the vial with a syringe (pay attention to the negative pressure in the vial, and the syringe must accurately draw 2 mL of PBS). The final concentration is 2 mg / mL, 4°C, overnight (preparation time: 23:37, November 22, 2021, and the sample production date is August 14, 2021).

[0123] Note: For protein samples, try testing samples with an A below 2 (A: UV absorbance). Keep the HT (instrument detection voltage) between 170 and 700. If it is >700, the test results may be inaccurate. Reduce the sample concentration, use a short-wavelength cuvette, or change the solvent. The recommended maximum concentration is 1-0.5 mg / mL, and it is recommended to set it according to the instrument sensitivity.

[0124] Sample testing:

[0125] Gradient dilution sample preparation: 1. Use an ice box ice maker to take ice, and perform a two-fold gradient dilution of the overnight sample at 4°C (note to change the pipette tip), take 500μl of the overnight sample, and dilute it with PBS to concentrations of 1.0, 0.5, 0.25, and 0.125mg / mL respectively

[0126] 2) Instrument (Circular Dichroism Spectrometer, JASCO J-815; JASCO) Parameter Setting

[0127] Band width: 1.0nm

[0128] Step: 1.0nm

[0129] Measurement range: 190-260nm

[0130] Time per point: 1s

[0131] Scanning speed: 50nm / min

[0132] Repeats: 3 times

[0133] Measurement temperature: 4°C

[0134] 3) Scanning of standard CD

[0135] The scanning wavelength was set to 190-260 nm for blank buffer baseline test, and the circular dichroism absorption of the sample solution PBS in the range of 190-260 nm was collected.

[0136] 4) Scanning spectrum processing.

[0137] Figure 4 shows the circular dichroism spectrum of recombinant humanized collagen C17T15. Recombinant humanized collagen C17T15 has a negative peak near 195 nm and a positive peak near 221 nm, indicating that under these conditions, the protein has a triple helical structure.

[0138] Example 4: CCK8 assay

[0139] 1. Digest, centrifuge, and count the HeLa cells cultured the day before.

[0140] 2. Inoculate 100 μL of 3-7k Hela cells / well on a 96-well plate and culture at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0141] 3. Observe the cell growth status and density under a microscope, and select wells with good growth status, uniform cell distribution and density for experiments.

[0142] 4. Prepare C17T15 sample solutions of different concentration gradients. Add three replicates of each concentration to a 96-well plate and incubate at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0143] 5. Add 50 μL of CCK-8 solution diluted according to the kit instructions (Tongren Chemical Research Institute; Cell counting KIT-8, YZ-CK04) to each well.

[0144] 6. Incubate at 37°C, 5% CO2, and 90% humidity for 0.5 hours.

[0145] 7. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0146] 8. Process and analyze the results using Graphpad Prism.

[0147] The results are shown in FIG5 : the constructed recombinant humanized collagen type XVII showed no obvious cytotoxicity to human cervical cancer cell Hela cells at a concentration below 250 μg / mL, and can be safely used in humans.

[0148] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Collagen, which comprises a plurality of repeating units, and each repeating unit comprises an amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO:1 are mutated; the number of repeating units is 10-20, 12-18 or 13-16; each repeating unit is directly connected or connected through a linker of one amino acid or multiple amino acids; Preferably, the mutation is substitution, insertion, deletion or addition; preferably, the substitution is a conservative amino acid substitution; Preferably, the collagen is derived from human; Preferably, the collagen has a triple helix structure or is collagen in the form of a triple helix structure; Preferably, the collagen has a cell adhesion effect; Preferably, the collagen is recombinant collagen, recombinant humanized collagen or recombinant humanized type XVII collagen.

2. The collagen according to claim 1, which comprises the following amino acid sequences: (1) The amino acid sequence shown in SEQ ID NO:2; (2) An amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2; or (3) An amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO:2 are mutated; preferably, the mutation is substitution, insertion, deletion or addition; preferably, the substitution is a conservative amino acid substitution.

3. Nucleic acid, which encodes the collagen according to claim 1 or 2; preferably, the nucleic acid has a nucleotide sequence shown in SEQ ID NO:

3.

4. Vector, which comprises the nucleic acid according to claim 3, optionally, the vector comprises nucleotides encoding a purification tag, nucleotides encoding a leader and / or regulatory elements; Preferably, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag; Preferably, the regulatory elements are selected from promoters, terminators and / or enhancers.

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

6. A method for producing collagen, which comprises: (1) Culturing the host cell according to claim 5 under suitable culture conditions; (2) Harvesting the host cell and / or the culture medium containing collagen; and (3) Purifying the collagen, for example, including (1) roughly purifying the collagen on a Ni affinity chromatography column; (2) adding a collagen tool enzyme for digestion; and / or (3) highly purifying the collagen on an ion exchange column.

7. A composition comprising the collagen according to claim 1 or 2, the nucleic acid according to claim 3, the carrier according to claim 4, and / or the host cell according to claim 5; Preferably, the composition is a pharmaceutical composition, a food composition or a cosmetic composition, Preferably, the composition is one or more of a biological dressing, a human biomimetic material, a plastic and aesthetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material, a blood vessel repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient and a food additive; Preferably, the composition comprises a pharmaceutically and / or cosmetically acceptable carrier; Preferably, the composition is a solid, liquid or gel composition; Preferably, the composition is a composition for oral and / or topical administration, preferably a topical application composition; Preferably, the composition is a kit; Preferably, the composition is a liquid formulation comprising the collagen according to claim 1 or 2 and a pharmaceutically and / or cosmetically acceptable carrier; preferably, the carrier is a buffer, such as D-PBS buffer or PBS buffer.

8. A method for promoting cell adhesion or attachment, the method comprising contacting cells with the collagen according to claim 1 or 2 and / or the composition according to claim 7.

9. Use of the collagen according to claim 1 or 2, the nucleic acid according to claim 3, the carrier according to claim 4, the host cell according to claim 5 and / or the composition according to claim 7 in a biological dressing, a human biomimetic material, a coating material, an organoid culture material or a 3D printed artificial organ biomaterial, or in a plastic and aesthetic material, an organoid culture material, a cardiovascular stent material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material or a blood vessel repair and regeneration material.

10. Use of the collagen according to claim 1 or 2 and / or the composition according to claim 7 in the preparation of a drug or a kit for promoting cell adhesion or attachment.

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