Method for manufacturing a human structural material by biosynthesis

Abstract

The present invention provides a method for manufacturing a human structural material by biosynthesis. The polypeptide of the present invention has a structure in the (repeating unit) n or (repeating unit) n -including the structure of the C-terminal region, and the repeating unit contains the amino acid sequence shown in SEQ ID NO: 1. The recombinant humanized type V collagen obtained in the present invention has high activity in promoting cell adhesion, does not cause an immune response even when applied to the human body, and further, the manufacturing method has inventiveness, enabling mass production of recombinant humanized type V collagen, and can be widely used in the manufacture of human structural materials. Its application fields include the manufacture of advanced medical devices such as biological coatings, human biomimetic materials, cosmetic and plastic materials, organoid culture, cardiovascular stents, coatings, tissue injection filling, ophthalmic materials, obstetrics and gynecology biological materials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, and biological materials for 3D printed artificial organs.

Description

Technical Field

[0001] This application claims the priority of the invention named "Method for Manufacturing Human Structural Materials by Biosynthesis" with Chinese application number: 202210849498.3 filed on July 19, 2022, and the entire disclosure content thereof is incorporated herein by reference.

[0002] The present invention belongs to the field of synthetic biology technology and relates to a method for manufacturing human structural materials by biosynthesis.

Background Art

[0003] Human structural materials are mainly structural proteins containing collagen. Such proteins have an adhesion function and a support function for cells and tissues and are the main components of the extracellular matrix. Collagen is a type of protein widely distributed in the connective tissues of the human body and is the most abundant protein in the human body, accounting for 25% - 35% of the total protein. Currently, at least 28 subtypes of collagen have been found to be present in different tissues and organs. Among them, type V collagen belongs to fibrous collagen, always accompanied by the expression of type I collagen, but with a low content and unique physiological functions. For example, it can inhibit the adhesion and proliferation of epidermal, endothelial, smooth muscle, and cancer cells, and can be used in the fields of medicine, materials, and biomedicine by combining with bioactive substances such as heparin, insulin, osteonectin, vascular endothelial inhibitor, macrophage colony-stimulating factor, etc.

[0004] In recent years, crude products of type V collagen have been mainly extracted from multiple types of aquatic products and tissues such as placenta, kidneys, etc. However, type V collagen has a low content in the body, is a minor collagen, and the extraction process is complex. At the same time, the immune response derived from animals is also a factor that restricts the application of collagen. With the growth of the collagen industry in China, the utilization of the biosynthetic pathway for obtaining collagen has become increasingly mature, especially humanized collagen is at the world's leading level. In 2021, the National Medical Products Administration named and classified collagen produced by biosynthesis. Among them, recombinant humanized collagen is a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination of fragments containing the function of human collagen.

[0005] Type V collagen was first discovered in the human placenta and skin, contains three different α-chains with different strengths and mobilities, and forms four different subtypes in tissues, namely, α1α1α2(V), α1α2α3(V), α3α3α3(V), and a mixture of type V collagen α-chains and type XI α-chains. The three different α-chains form three string-like procollagen molecules, which become elongated collagen fibers through extracellular enzymatic degradation. The elongated fibers cross-link with each other in the space around the cells, forming a high-strength mature type V collagen fiber network after cross-linking, or forming heterotrimers with other types of collagen such as type XI collagen. It is distributed in sites such as the cornea, skin, ligament, bone, tendon, and muscle. Type V collagen has the common structural characteristics of fibrillar collagen, and in the central region, there is a continuous sequence of more than 1000 Gly-Xaa-Yaa. Here, Xaa-Yaa is any amino acid or imino acid residue. Structurally speaking, the natural type V collagen in the human body has a very complex structure, so it is difficult to express humanized collagen by conventional means and to prepare it in large quantities.

[0006] Currently, the production of type V collagen is mainly obtained by treating animal-derived tissues through pepsin and extracting collagen derivatives. However, the collagen extracted by these methods has lost its original biological activity and cannot exert its original functions. With the development of modern biotechnology, people can prepare recombinant humanized collagen in the expression systems of animals, plants, and microorganisms through gene transfer technology, thus solving the drawbacks of the enzymatic degradation method. However, currently, no success has been achieved in obtaining recombinant humanized type V collagen. Therefore, there is an urgent need for a biosynthesis method of recombinant type V humanized collagen so that it can be widely used as a human body structural material.

Summary of the Invention

Means for Solving the Problems

[0007] In consideration of the technical problems existing in the prior art, the inventors have designed for the first time the screening of the functional regions of recombinant humanized type V collagen and the protein synthesis process. The inventors have found the amino acid sequence of the core region of type V collagen (SEQ ID NO: 1: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv), and using this core region as a repeating unit, a polypeptide containing multiple repeating units has been constructed. The inventors have found that the constructed polypeptide has the activity of promoting cell adhesion. The inventors have found that one or more amino acids can be extended as repeating units at the N-terminus and / or C-terminus of the core region, and the constructed polypeptide containing multiple repeating units also has the activity of promoting cell adhesion. Furthermore, outside the repeating unit, the polypeptide may contain a peptide segment of a certain length outside the repeating unit (for example, at the N-terminus and / or C-terminus of the polypeptide composed of repeating units). This peptide segment may be a continuous amino acid segment starting from the first position of the repeating unit.

[0008] In one aspect, the present invention is (repeating unit) n or (repeating unit) nProvided is a polypeptide comprising the structure of the C-terminal region, wherein the repeating unit comprises the amino acid sequence shown in SEQ ID NO: 1, or the repeating unit comprises the amino acid sequence shown in SEQ ID NO: 1 and additional amino acid residues at the N-terminus and / or C-terminus of SEQ ID NO: 1, and the number of the additional amino acid residues is 1 to 50. The polypeptide of the present invention is recombinant humanized type V collagen.

[0009] In one embodiment, each repeating unit is directly linked, and the number n of the repeating units is 4 to 20, and the C-terminal region is a continuous amino acid segment starting from the first position of the repeating unit.

[0010] In one embodiment, the additional amino acid residues are at the C-terminus of SEQ ID NO: 1 and are the amino acid sequence shown in SEQ ID NO: 7 (galglk) or a continuous amino acid segment of the amino acid sequence. For example, the additional amino acid residues are a continuous amino acid segment starting from the first position of the amino acid sequence shown in SEQ ID NO: 7.

[0011] In one embodiment, the repeating unit comprises any one of the amino acid sequences of SEQ ID NOs: 2, 3, and 8 to 11.

[0012] In one embodiment, the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 4 to 6.

[0013] In another aspect, the present invention provides a nucleic acid comprising the nucleotide of the polypeptide of the present invention.

[0014] In one embodiment, the nucleic acid further comprises a nucleotide encoding a purification tag such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag.

[0015] In one embodiment, the nucleic acid further comprises a nucleotide encoding a leader sequence.

[0016] In another aspect, the present invention provides a vector comprising the nucleic acid according to the present invention.

[0017] In one embodiment, the vector includes expression control elements such as a promoter, a terminator, and / or an enhancer operably linked to a nucleic acid.

[0018] In another aspect, the present invention provides a host cell comprising the nucleic acid or vector of the present invention. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell.

[0019] In another aspect, the present invention provides for the production of the polypeptide of the present invention comprising the following.

[0020] (1) Culturing the host cell of the present invention under appropriate culture conditions;

[0021] (2) Recovering the host cell and / or the medium containing the polypeptide; and

[0022] (3) Purifying the fusion protein or the trimeric fusion protein.

[0023] In another aspect, the present invention provides a composition comprising the polypeptide, nucleic acid, vector, and / or host cell of the present invention.

[0024] In one embodiment, the composition is one or more of a biological coating material, a human biomimetic material, a cosmetic and plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filler, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration material, a liver tissue material, and a blood vessel repair and regeneration material, a biological material for a 3D printed artificial organ, a cosmetic raw material, a pharmaceutical adjuvant, and a food additive.

[0025] In another aspect, the present invention provides for the use of the polypeptide, nucleic acid, vector, host cell, and / or composition of the present invention in promoting cell adhesion in vitro or in the preparation of a drug for promoting cell adhesion.

[0026] In another aspect, the present invention provides for the use of the polypeptide, nucleic acid, vector, host cell and / or composition of the present invention in the manufacture of advanced medical devices such as biological coating materials, human biomimetic materials, cosmetic and plastic materials, organoid culture, cardiovascular stents, coatings, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, biomaterials for 3D printed artificial organs, as well as high-end cosmetic raw materials, advanced pharmaceutical adjuvants, and food additives.

[0027] The present invention discloses the specific process of screening and synthesis of the functional regions of recombinant humanized type V collagen, which can be used in the manufacture of human structural materials. The present invention belongs to the technical field of synthetic biology. The present invention discloses the specific process of screening and synthesis of the functional regions of recombinant humanized type V collagen, which can be used in the manufacture of human structural materials. The present invention belongs to the technical field of synthetic biology. The biosynthetic method for preparing recombinant humanized type V collagen in the present invention includes: (1) screening of functional regions and construction of strains; (2) biological fermentation, induction and expression; (3) purification of humanized type V collagen and optional enzymatic cleavage. The amino acid sequence of the recombinant humanized collagen produced by the present invention is derived from the functional regions of human natural type V collagen, and includes proteins in which the functional regions and similar functional regions, amino acid sequences are mutated and modified respectively. The recombinant humanized type V collagen produced by the present invention has high activity in promoting cell adhesion, is applied so as not to cause an immune response in the human body, and its preparation method is novel. Recombinant humanized type V collagen can be obtained in large quantities and is widely used in the preparation of human structural materials. Its application fields include the manufacture of advanced medical devices such as biological coating materials, human biomimetic materials, cosmetic and plastic materials, organoid culture, cardiovascular stents, coatings, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, biomaterials for 3D printed artificial organs, as well as high-end cosmetic raw materials, advanced pharmaceutical adjuvants, food additives, etc.

[0028] The present invention provides the following. <1>Regarding the current state of research, the present invention provides a method for producing recombinant humanized type V collagen by biosynthesis, that is, a method for producing a human structural material. Specifically, it includes: (1) screening of functional regions and construction of strains; (2) large-scale bioreactor culture and induction of protein expression; (3) purification of humanized type V collagen and optional enzymatic cleavage. <2>According to <1>, the screening of functional regions and construction of strains can be carried out as follows: (1) screening a large-scale functional region to obtain the functional region of the target gene; (2) inserting the obtained functional region of the target gene into a PET-32a expression vector to obtain a recombinant expression plasmid; (3) introducing the recombinant expression plasmid into competent cells of Escherichia coli BL21(DE3), and screening to obtain positive gene recombinant Escherichia coli. <3>According to <1>, large-scale bioreactor culture can be carried out by adding the positive gene recombinant Escherichia coli obtained by screening into a flask containing an antibiotic stock solution and culturing it in a constant temperature shaker at 220 rpm and 37 °C for 7 hours. <4>According to <1>, induction of protein expression can be carried out as follows: (1) cooling the cultured flask to 16 °C; (2) adding an IPTG stock solution to induce expression; (3) putting the induced bacterial solution into a centrifuge bottle and centrifuging it at 8000 rpm and 4 °C for 10 minutes to collect the bacterial cells. <5>According to <1>, purification of humanized type V collagen and optional enzymatic cleavage can be carried out as follows: (1) roughly purifying humanized type V collagen using a Ni affinity chromatography column; (2) adding TEV enzyme at a certain ratio to perform enzymatic cleavage; (3) re-purifying humanized type V collagen using an ion exchange column. According to <6><2>, the screened functional regions are as follows: (1) C5V3G1 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk, (2) C5V3G2 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal,(3) C5V3G3 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv. <7>The amino acid sequence of the recombinant humanized type V collagen produced in the present invention is derived from the functional region of human native type V collagen and includes proteins in which the functional region and similar functional regions, and amino acids are mutated and modified, respectively. <8>The recombinant humanized type V collagen produced in the present invention includes, in its application fields, production of advanced medical devices such as biological coatings, human biomimetic materials, cosmetic and plastic surgery materials, organoid culture, cardiovascular stents, coatings, myocardial repair, tissue injection filling, ophthalmic materials, obstetrics and gynecology biological materials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, biological materials for 3D printed artificial organs, tumor prevention, bone repair materials, skin repair materials, kidney tissue repair, and pancreas repair; high-grade cosmetic raw materials, advanced pharmaceutical adjuvants, food additives, etc.

[0029] Compared with the prior art, the present invention has the following advantages.

[0030] <1>The present invention provides the core functional region and amino acid sequence of recombinant humanized type V collagen.

[0031] <2>The present invention has succeeded in the biosynthesis of recombinant humanized type V collagen for the first time.

[0032] <3>The recombinant humanized type V collagen produced by the present invention has a good cell adhesion effect and does not cause an immune response when applied to the human body.

[0033] <4>Its production method is simple, and recombinant humanized type V collagen can be obtained on a large scale.

Brief Description of the Drawings

[0034]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, hereinafter, 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 only some, rather than all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments described in this specification without any inventive effort shall fall within the protection scope of the present invention.

[0036] As used herein, a polypeptide means a plurality of amino acid residues linked by peptide bonds. In this specification, the polypeptide contains a plurality of repeating units derived from human type V collagen (GenBank: KAI4009058.1). The polypeptide may contain (repeating unit) n or (repeating unit) n - may include the structure of the C-terminal region, the repeating unit contains the amino acid sequence shown in SEQ ID NO: 1, or contains the amino acid sequence shown in SEQ ID NO: 1 and additional amino acid residues at the N-terminus and / or C-terminus of SEQ ID NO: 1, and the number of said additional amino acid residues is 1 to 50. For example, the number of said additional amino acid residues may be 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 or 49. Said additional amino acid residues can be derived from human type V collagen. For example, said additional amino acid residues may be a continuous amino acid segment directly adjacent to the repeating unit in situ of natural human type V collagen. Said additional amino acid residues may be the amino acid sequence shown in SEQ ID NO: 7 or a continuous amino acid segment of said amino acid sequence. Preferably, said additional amino acid residues are a continuous amino acid segment starting from the first position of the amino acid sequence shown in SEQ ID NO: 7. In this specification, said additional amino acid residues may be located at the C-terminus.

[0037] In this specification, each repeating unit may be directly connected or may have one or more amino acid residues as a spacer. The number n of repeating units may be 4 to 20, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. The polypeptide may include a C-terminal region. This C-terminal region may be a continuous amino acid segment starting from the first position of the repeating unit. The repeating unit may contain the amino acid sequence shown in either SEQ ID NO: 2 or 3.

[0038] As used herein, when the expression "segment" is used in relation to amino acids, it refers to a part of a sequence that is smaller than a specific sequence. For example, an amino acid segment in SEQ ID NO:7 means a subsequence of any length that is shorter than the length of the sequence shown in SEQ ID NO:7. A "continuous amino acid segment" refers to an amino acid segment consisting of directly adjacent amino acids in a sequence.

[0039] As used herein, with respect to a polypeptide or a specific amino acid sequence, "C-terminus" and "N-terminus" mean positions relative to the polypeptide or the specific amino acid sequence, specifically, they mean being located at the end having a carboxyl group or the end having an amino group of the polypeptide or the specific amino acid sequence.

[0040] As used herein, when referring to a polypeptide or a specific amino acid sequence, its position is described relative to the C-terminus of the polypeptide or the specific amino acid sequence. For example, when it is said "from the 1st position of the amino acid sequence (galglk) shown in SEQ ID NO:7", the 1st amino acid refers to G.

[0041] In the present specification, the repeating unit of the present invention may include the following sequences or may be the following sequences. gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv (SEQ ID NO:1) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal (SEQ ID NO:2) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk (SEQ ID NO:3) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvg (SEQ ID NO:8) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvga (SEQ ID NO:9) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalg (SEQ ID NO:10) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgl (SEQ ID NO: 11)

[0042] As used herein, "nucleic acid" refers to a plurality of nucleotides connected via a connection between nucleotides. The connection between nucleotides may be, for example, a phosphodiester bond. The nucleic acids herein can include polynucleotides encoding the polypeptides of the present invention. To facilitate post-treatment of the polypeptide, the nucleic acids of the present invention may further include nucleotides encoding a purification tag such as a His tag, GST tag, MBP tag, SUMO tag or NusA tag, and nucleotides encoding a leader sequence as needed.

[0043] As used herein, the term "vector" is a nucleic acid carrier into which a polynucleotide can be inserted. When a protein encoded by the polynucleotide inserted into the vector can be expressed, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, and the carried genetic factor can be 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); phages, such as λ phage or M13 phage; and animal viruses; etc. The vector may include multiple types of expression control elements. The aforementioned expression control elements include, but are not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector may further include an origin of replication site. The vector may include the nucleic acids of the present invention to facilitate expression when introduced into a cell. The vector may include expression control elements operably connected to the nucleic acid, such as a promoter, a terminator and / or an enhancer.

[0044] As used herein, the term "host cell" refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection by viral vectors, transformation by plasmid vectors, and introduction of naked DNA at high speed by electroporation, lipofection, and particle gun. The host cell may be a eukaryotic cell or a prokaryotic cell. For example, the eukaryotic cell may be a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell may be an Escherichia coli cell.

[0045] In the present specification, each part of the polypeptide of the present invention, for example, the repeating unit or the C-terminal region, may have certain mutations. For example, one or more of these amino acid sequences may have substitutions, deletions, additions, or insertions of amino acid residues. That is, the present invention can use variants of each part such as repeating unit variants or C-terminal region variants as long as they retain the activity of promoting cell adhesion. Specifically, the variant can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a specific sequence. The specific sequence may be any sequence in the present invention, for example, SEQ ID NOs: 1 to 6, but these variants preferably retain the core sequence identified in the present invention.

[0046] The polypeptide of the present invention can be prepared by any suitable method, for example, it can be prepared by synthesis. Preferably, the polypeptide of the present invention can be prepared recombinantly. The production method may include one or more of the following steps: (1) culturing the host cell according to claim 8 under appropriate culture conditions; (2) obtaining the host cell and / or medium containing the polypeptide; and (3) purifying the fusion protein or trimeric fusion protein. More specifically, the sequence encoding the polypeptide of the present invention is inserted into an appropriate expression vector, such as PET-28A, to obtain a recombinant expression vector. The recombinant expression plasmid is introduced into competent cells of Escherichia coli BL21(DE3), and positive gene-recombinant Escherichia coli is obtained by screening. Then, the obtained positive gene-recombinant Escherichia coli can be subjected to large-scale biological fermentation (for example, cultured in a constant-temperature shaker at 220 rpm and 37 °C for 7 hours). When culturing to an appropriate cell density, protein expression can be induced intracellularly. For example, in the case of gene-recombinant Escherichia coli, (1) the culture flask after culturing is cooled to 16 °C, (2) an IPTG stock solution is added to induce expression, (3) the bacterial solution after expression induction is put into a centrifuge bottle, centrifuged at 8000 rpm and 4 °C for 10 minutes, and then the bacterial cells are collected. After lysing the bacterial cells (for example, by high-pressure homogenization) to obtain a cell lysate, centrifugation is performed to obtain a supernatant. The supernatant is purified to obtain a purified polypeptide. For example, the purification process may include one or more of the following steps: (1) a step of roughly purifying the polypeptide using a Ni affinity chromatography column, (2) a step of adding TEV enzyme at a certain ratio for enzymatic cleavage, and (3) a step of re-purifying the polypeptide using an ion exchange column.

[0047] The polypeptide, nucleic acid, vector and / or host cell of the present invention can be prepared as a composition or a kit. The composition or kit can include one or more of a bio-coating material, a human biomimetic material, a cosmetic and plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filler, 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 biomaterial for 3D printed artificial organs, a cosmetic raw material, a pharmaceutical adjuvant, and a food additive. The composition or kit can be one or more of a bio-coating material, a human biomimetic material, a cosmetic and plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filler, 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 biomaterial for 3D printed artificial organs, a cosmetic raw material, a pharmaceutical adjuvant, and a food additive. This composition or kit can be used to promote cell adhesion in vitro or in vivo.

Example

[0048] Hereinafter, the present invention will be further specifically described by way of examples. Those skilled in the art should understand that these examples are merely illustrative and not restrictive. The present invention is limited only by the claims.

[0049] Example 1: Construction and Expression of Recombinant Humanized Type V Collagen <1>Perform large-scale screening of functional regions to obtain the following target gene fragments of different recombinant humanized type V collagen: (1) C5V3G1 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk (C5V3G1 amino acid sequence: SEQ ID NO: 6; the repeating unit is SEQ ID NO: 3: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk);

[0050] (2) C5V3G2 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal (C5V3G2 amino acid sequence: SEQ ID NO: 5; the repeating unit is SEQ ID NO: 2: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal);

[0051] (3) C5V3G3 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv (C5V3G3 amino acid sequence: SEQ ID NO: 4; the repeating unit is SEQ ID NO: 1: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv).

[0052]

[0053] <2>The constructed expression plasmid was used to transform the competent cells of Escherichia coli BL21(DE3). Specifically, (1) The competent cells of Escherichia coli BL21(DE3) were taken out from the ultra-low temperature freezer, placed on ice, and when semi-melted, 2 μl of the plasmid to be transformed was taken and added to the competent cells of Escherichia coli BL21(DE3), and gently mixed 2 - 3 times. (2) The mixture was placed on ice for an ice bath for 30 minutes, given a heat shock in a 42 °C water bath for 45 - 90 seconds, taken out, and placed on ice for an ice bath for 2 minutes. (3) It was transferred to a biological safety cabinet, 700 μl of liquid LB medium was added, and cultured at 37 °C and 220 rpm for 60 minutes. (4) 200 μl of the bacterial solution was taken and evenly spread on an LB plate containing sodium ampicillin. (5) The plate was cultured in an incubator at 37 °C for 15 - 17 hours until colonies of uniform size were obtained.

[0054] <3>5 - 6 single colonies were picked from the transformed LB plate and transferred to a flask containing the antibiotic stock solution, and cultured in a constant temperature shaker at 220 rpm and 37 °C for 7 hours. After culturing, the flask was cooled to 16 °C, IPTG was added for expression induction, and after a certain period of time, the bacterial solution was dispensed into a centrifuge bottle, centrifuged at 8000 rpm and 4 °C for 10 minutes, the bacterial cells were collected, the weight of the bacterial cells was recorded, sampled, and electrophoresis was performed (refer to the "Bacterial cells" lanes in Figure 1 and Figure 2).

[0055] <4>The collected bacterial cells were resuspended in an appropriate buffer, the bacterial solution was cooled to 15 °C or below, homogenization and high-pressure homogenization were performed twice, and after completion, the bacterial solution was collected. The homogenized bacterial solution was dispensed into a centrifuge bottle, centrifuged at 17000 rpm and 4 °C for 30 minutes, the supernatant was collected, and the supernatant and precipitate were sampled and electrophoresis was performed (refer to Figure 1 and Figure 2, "Homogeneous" is the electrophoresis diagram of the bacterial solution after performing high-pressure homogenization twice, and "Supernatant" and "Precipitate" are the electrophoresis diagrams of the supernatant and precipitate respectively).

[0056] <5>The purification and enzymatic cleavage of recombinant humanized type V collagen are specifically as follows: (1) Crude purification: a. Wash the column with water; b. Equilibrate the column; c. Load the sample: Put the centrifuged supernatant into the column, and sample the flow-through until all the liquid has flowed out and perform electrophoresis (refer to the "flow-through" lanes in Figures 1 and 2); d. Wash the impurities: Add 25 mL of the washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), and sample the flow-through until all the liquid has flowed out and perform electrophoresis (refer to the "washing" lanes in Figures 1 and 2); e. Recover the target protein: Add 20 mL of the eluent, collect the flow-through solution, and obtain the target protein containing the His tag. Detect the protein concentration by ultraviolet-visible spectrophotometry, calculate the protein concentration using the following formula (C (mg / ml) = A280 × dilution factor × extinction coefficient), and perform electrophoresis (refer to the "elution" lanes in Figures 1 and 2); f. Wash the column with 1M imidazole buffer; g. Wash the column with purified water. (2) Enzymatic cleavage: The ratio of the total amount of protein to the total amount of TEV enzyme is 20:1. Add TEV enzyme and perform enzymatic cleavage at 16°C for 2 hours, and sample and perform electrophoresis. Put the enzymatically cleaved protein solution into a dialysis bag, dialyze at 4°C for 2 hours, and then transfer it to a new dialysis solution and dialyze at 4°C overnight (refer to Figure 3 for the electrophoresis diagram after liquid exchange). (3) Repurification: a. Equilibrate the column: Use Solution A (20 mM Tris, 20 mM sodium chloride) to equilibrate the column at a flow rate of 10 ml / min; b. Load the sample: Set the flow rate to 5 mL / min, load the sample, collect the flow-through (denoted as FL1, refer to Figure 3), and obtain recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3. Perform electrophoresis as shown in Figure 3; c. Elution: Set 100% Solution B (20 mM Tris, 1M sodium chloride, pH 8.0) for 3 CV elution, peak out and collect, and perform electrophoresis (for the Solution B eluate, refer to the "Solution B elution" lane in Figure 3); d. Wash the column; e. Store the protein at 4°C.Figure 3 shows that the apparent molecular weights of recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3 are 32 kDa, 29 kDa, and 34 kDa, respectively, which are consistent with the predicted molecular weights.

[0057] Example 2: Detection of Biological Activity of Recombinant Humanized Type V Collagen Regarding the method for detecting the activity of collagen, reference can be made to 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 can be exemplified as follows.

[0058] (1) By the ultraviolet absorption method, the concentrations of the measured protein samples such as bovine type I collagen (National Institutes for Food and Drug Control, number: 380002; 0.5 mg / mL, positive control well), recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3 provided in the present invention were detected.

[0059] Specifically, the absorption of ultraviolet light at 215 nm and 225 nm of the sample was measured respectively, and the protein concentration was determined using the experimental formula C (μg / M) = 144×(A215 - A225) (it is necessary to detect when A215 < 1.5). The principle of this method is to measure the characteristic absorption of peptide bonds in far ultraviolet light, which is less affected by the content of chromophores, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not color with Coomassie Brilliant Blue (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43 - 45.). After detecting the protein concentration, all measured protein concentrations were adjusted to 0.25 mg / mL, 0.5 mg / mL, or 1 mg / mL with PBS.

[0060] (2) 100 μL of various protein solutions and blank PBS solution control (refer to the D - PBS group in Figures 4 - 6) were added to a 96 - well plate and left at room temperature for 60 minutes.

[0061] (3) 10 5 cultured 3T3 cells in good condition were placed in each well and incubated at 37 °C for 60 minutes.

[0062] (4) Each well was washed 4 times with PBS.

[0063] (5) The absorbance at OD492 nm was detected using an LDH detection kit (Roche, 04744926001). From the value of the blank control, the cell adhesion rate can be calculated. The calculation formula is as follows:

Number

[0064] As shown in Figs. 4, 5, and 6, the recombinant humanized type V collagen of the present invention was found to have better biological adhesion activity than bovine type I collagen (BColI group, 0.5 mg / ml). Fig. 4 shows the relative cell adhesion activity of C5V3G1 against bovine type I collagen, and it was revealed that C5V3G1 has cell adhesion activity and has higher cell adhesion activity than bovine type I collagen at concentrations of 0.25, 0.5, and 1 mg / ml. Fig. 5 shows the relative cell adhesion activity of C5V3G2 against bovine type I collagen, and it was revealed that C5V3G2 has better cell adhesion activity at concentrations of 0.25 mg / mL or higher. Fig. 6 shows the relative cell adhesion activity of C5V3G3 against bovine type I collagen, and it was revealed that C5V3G3 has cell adhesion activity and has higher cell adhesion activity than bovine type I collagen at a concentration of 0.5 mg / ml.

[0065] Example 3: Detection of Recombinant Humanized Type V Collagen by Mass Spectrum [Experimental Method] [Number]

[0066] The protein sample was reduced with DTT, alkylated with iodoacetamide, and then trypsin was added for overnight enzymatic digestion. The digested peptide segments were further desalted with C18 ZipTip and then mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) and spotted. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometer MALDI-TOF / TOF Ulraflextreme TM, analyzed by Brucker, Germany (the peptide fingerprinting technique can refer to Protein J. 2016;35:212-7). The data search was processed on the MS / MS Ion Search screen from the local mascot site. The protein identification results were obtained from the primary mass spectrometry of enzymatically cleaved peptide segments. Detection parameters: Hydrolyzed with trypsin, with two missed cleavage sites provided. The alkylation of cysteine was set as a fixed modification. The oxidation of methionine is a variable modification. The database used for identification was NCBprot.

[0067]

Table 1

[0068] The coverage rate of the detected polypeptide segments was 100%, consistent with the theoretical sequence, and the detection results were highly reliable.

[0069]

Table 2

[0070] The detected polypeptide segments had a coverage rate of 83% compared to the theoretical sequence, and the detection results were highly reliable.

[0071]

Table 3

[0072] The detected polypeptide segments had a coverage rate of 96.11% compared to the theoretical sequence, and the detection results were highly reliable.

[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any modifications, improvements, substitutions, combinations, simplifications, etc., as long as they do not depart from the spirit and principle of the present invention, are all equivalent substitution methods and are all included in the protection scope of the present invention.

Claims

1. (Repeating unit) ｎ or (Repeating unit) ｎ -including the structure of the C-terminal region, The repeating unit contains the amino acid sequence shown in SEQ ID NO: 1, or the repeating unit contains the amino acid sequence shown in SEQ ID NO: 1 and additional amino acid residues at the N-terminus and / or C-terminus of SEQ ID NO: 1, and the number of the additional amino acid residues is 1 to 50, each repeating unit is directly linked, and the number n of the repeating units is 4 to 20, and the C-terminal region is a continuous amino acid segment starting from the first position of the repeating unit, a polypeptide, preferably recombinant humanized type V collagen.

2. the additional amino acid residue is at the C-terminus of SEQ ID NO: 1, and the additional amino acid residue is the amino acid sequence shown in SEQ ID NO: 7 or a continuous amino acid segment of the amino acid sequence thereof, the polypeptide according to claim 1.

3. the additional amino acid residue is a continuous amino acid segment starting from the first position of the amino acid sequence shown in SEQ ID NO: 7, the polypeptide according to claim 2.

4. the repeating unit contains any one of the amino acid sequences of SEQ ID NOs: 2, 3, and 8 to 11, the polypeptide according to any one of claims 1 to 3.

5. contains the amino acid sequence selected from SEQ ID NOs: 4 to 6, the polypeptide according to any one of claims 1 to 4.

6. contains a nucleotide encoding the polypeptide according to any one of claims 1 to 5, optionally, further contains a nucleotide encoding a purification tag such as His tag, GST tag, MBP tag, SUMO tag or NusA tag, optionally, further contains a nucleotide encoding a leader sequence, a nucleic acid.

7. contains the nucleic acid according to claim 6, optionally, contains an expression control element such as a promoter, a terminator and / or an enhancer operably linked to the nucleic acid, a vector.

8. contains the nucleic acid according to claim 6 or the vector according to claim 7, preferably, 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 Escherichia coli such as Escherichia coli BL21, a host cell.

9. a method for producing the polypeptide according to any one of claims 1 to 5, comprising: (1) culturing the host cell according to claim 8 under appropriate culture conditions, (2) recovering the host cell and / or the medium containing the polypeptide, and (3) Purifying the fusion protein, for example, 1) roughly purifying the polypeptide with a Ni affinity chromatography column; 2) adding TEV enzyme at a certain ratio and performing enzymatic cleavage; 3) re-purifying the polypeptide with an ion exchange column, A method comprising the above. **Claim 10** A composition comprising the polypeptide according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7 and / or the host cell according to claim 8. **Claim 11** The composition according to claim 10, which is one or more of a biological coating material, a human biomimetic material, a cosmetic and plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a biological material for a 3D printed artificial organ, a cosmetic raw material, a pharmaceutical adjuvant, and a food additive. **Claim 12** Use of the polypeptide according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, the host cell according to claim 8 and / or the composition according to claim 10 for promoting cell adhesion in vitro or in the preparation of a product or kit for promoting cell adhesion. **Claim 13** Use of the polypeptide according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, the host cell according to claim 8 and / or the composition according to claim 10 in the manufacture of advanced medical devices such as a biological coating material, a human biomimetic material, a cosmetic and plastic surgery material, an organoid culture, a cardiovascular stent, a coating, a tissue injection filling, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration, a liver tissue and a blood vessel repair and regeneration, a biological material for a 3D printed artificial organ, in high-grade cosmetic raw materials and advanced pharmaceutical adjuvants, and in food additives.

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