Recombinant humanized type iii triple helical collagen, preparation method therefor, and use thereof

Abstract

Provided are a use of a recombinant humanized type III triple helical collagen and a preparation method therefor. Specifically, provided is a recombinant collagen monomer. The recombinant collagen monomer comprises at least one collagen active fragment, the collagen active fragment comprises at least three GER sites and / or GEK sites and / or RGD sites and / or GEN, and three adhesion sites are the same or different. The collagen active fragment is from the 168th amino acid to the 1196th amino acid of collagen.

Description

Recombinant humanized type III triple-helical collagen and its preparation method and application

[0001] Priority information

[0002] This application claims priority and benefits of patent application No. 2023110945209 filed with the State Intellectual Property Office of China on August 28, 2023, and the entire text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, and in particular to a recombinant humanized type III triple-helical collagen protein and a preparation method and application thereof. Background Art

[0004] Natural collagen (native collagen) is the most abundant macromolecular protein in living organisms, accounting for approximately 20-30% of the total protein content. Approximately 28 types of natural collagen have been discovered, and they are widely distributed in the connective tissues of skin, bones, tendons, and various organs. They participate in a variety of life activities, including wound repair, skin aging, blood coagulation, and joint lubrication. As an important biomaterial, collagen has extensive applications in medicine, cosmetology, and the food industry.

[0005] Natural type I and type III collagen are fibrous collagens with a complex, multi-layered structure. They have a large molecular weight, and the amino acid sequence of the helical domain within the molecule follows a repeating Gly-XY sequence, with Y typically being a hydroxylated proline. Three homologous or heterologous protein molecules are arranged into a triple helical structure through intermolecular forces such as hydrogen bonds and electrostatic forces, which then stack to form a higher-order fibrous structure.

[0006] Currently, collagen on the market primarily comes from animals such as pigs, cattle, and fish products. Animal-derived collagen is primarily extracted through acid and alkaline hydrolysis and enzymatic extraction processes. However, animal-derived collagen has the following disadvantages: 1) Because animal-derived collagen differs from human-derived collagen in amino acid sequence, it is immunogenic and therefore carries the risk of allergic reactions; 2) animal-derived collagen may contain viruses, such as mad cow disease prions; 3) the collagen structure is damaged to a certain extent during the extraction process, resulting in a reduction in its original biological activity; and 4) there are religious and ethical issues.

[0007] Therefore, there is an urgent need to develop a new method for preparing collagen to overcome the above-mentioned problems.

[0008] Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0010] With the recent development of synthetic biology, researchers have utilized genetic recombinant expression technology to mass-produce recombinant human collagen using microorganisms (such as Escherichia coli and yeast) as cell factories. Compared to traditional animal collagen extraction processes, recombinant collagen production offers the following advantages: 1) The amino acid sequence of recombinant human collagen is identical to natural human collagen, thus eliminating the safety risk of animal immunogenicity; 2) it avoids the spread of animal-derived viruses; and 3) the microbial fermentation process is low-cost and has a short production cycle, making it suitable for large-scale production. In recent years, the Pichia pastoris expression system has been widely used for recombinant collagen production. The inventors discovered that the Pichia pastoris expression system combines the advantages of both prokaryotic and eukaryotic expression systems, such as ease of operation, high expression yields, and low cost, making it suitable for large-scale industrial production. Furthermore, it allows for a certain degree of post-translational modification of exogenous proteins. Through genetic engineering, the inventors designed and modified the Pichia pastoris expression system and discovered that yeast-expressed humanized collagen has the ability to form a triple-helical conformation. Humanized collagen with a triple-helical conformation and biological activity is a high-quality biomaterial that closely resembles natural collagen. In addition, the inventors also found that Pichia pastoris has a high protein secretion efficiency and low self-protein secretion, so it can achieve high-level secretion of exogenous proteins and easy purification, and can avoid the toxic side effects caused by the accumulation of fermentation products.

[0011] Therefore, in the first aspect of the present invention, the present invention proposes a recombinant collagen monomer. According to an embodiment of the present invention, the recombinant collagen monomer includes at least one collagen active fragment, and the collagen active fragment includes at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites. After continuous experiments, the inventors found that the relevant peptide segments including at least 3 adhesion-related sites GER and / or GEK and / or RGD sites and / or GEN sites in natural collagen are collagen adhesion active sites. When the natural collagen is artificially broken, the peptide segments obtained only include at least 3 adhesion-related sites GER and / or GEK and / or RGD sites and / or GEN sites. They have adhesion activity or expression or secretion activity in recombinant cells.

[0012] According to an embodiment of the present invention, the recombinant collagen monomer further includes at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the collagen active fragment is derived from amino acids 168 to 1196 of collagen.

[0014] According to an embodiment of the present invention, the collagen active fragment includes three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0015] According to an embodiment of the present invention, the three GER sites and / or GEK sites and / or RGD sites and / or GEN sites are the same or different, for example, they may include three GER sites or three GEK sites or two GEK sites and one GER site.

[0016] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 1089 and 1172 of collagen. The inventors have discovered that within this range, the active collagen fragment exhibits the highest adhesion activity, expression, and secretion, laying the foundation for the subsequent preparation of recombinant collagen with high cell adhesion activity.

[0017] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 234 and 296 of collagen.

[0018] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 273 and 311 of collagen.

[0019] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 288 and 452 of collagen.

[0020] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 300 and 485 of collagen.

[0021] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 444 and 503 of collagen.

[0022] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 483 and 512 of collagen.

[0023] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 501 to 599 of collagen.

[0024] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 507 and 686 of collagen.

[0025] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 591 to 731 of collagen.

[0026] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 678 and 806 of collagen.

[0027] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 726 and 830 of collagen.

[0028] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 795 and 833 of collagen.

[0029] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 798 and 839 of collagen.

[0030] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 852 and 866 of collagen.

[0031] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 828 and 932 of collagen.

[0032] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 861 and 995 of collagen.

[0033] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 927 and 1094 of collagen.

[0034] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 987 and 1103 of collagen.

[0035] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 1089 to 1172 of collagen and / or a peptide segment between amino acids 234 to 296 and / or a peptide segment between amino acids 273 to 311 and / or a peptide segment between amino acids 288 to 452 and / or a peptide segment between amino acids 300 to 485 and / or a peptide segment between amino acids 444 to 503 and / or a peptide segment between amino acids 483 to 512 and / or a peptide segment between amino acids 501 to 599 and / or a peptide segment between amino acids 507 to 686. and / or a peptide segment between amino acid positions 591 and 731 and / or a peptide segment between amino acid positions 678 and 806 and / or a peptide segment between amino acid positions 726 and 830 and / or a peptide segment between amino acid positions 795 and 833 and / or a peptide segment between amino acid positions 798 and 839 and / or a peptide segment between amino acid positions 852 and 866 and / or a peptide segment between amino acid positions 828 and 932 and / or a peptide segment between amino acid positions 861 and 995 and / or a peptide segment between amino acid positions 927 and 1094 and / or a peptide segment between amino acid positions 987 and 1103.

[0036] According to an embodiment of the present invention, the collagen active fragment has an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0037] According to an embodiment of the present invention, 2 to 12 collagen active fragments are included.

[0038] According to an embodiment of the present invention, 6 collagen active fragments are included.

[0039] According to an embodiment of the present invention, the collagen active fragments are the same or different.

[0040] According to an embodiment of the present invention, the 2 to 12 collagen active fragments are directly or indirectly connected to each other.

[0041] According to an embodiment of the present invention, the present invention further comprises a stabilizing sequence connected to the C-terminus of the at least one collagen active fragment, thereby improving the stability of the collagen active monomer.

[0042] According to an embodiment of the present invention, the amino acid sequence of the stabilizing sequence is SEQ ID NO: 6.

[0043] According to an embodiment of the present invention, the recombinant collagen monomer has an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 25.

[0044] In its second aspect, the present invention provides a recombinant collagen. According to an embodiment of the present invention, the recombinant collagen comprises three recombinant collagen monomers as described in the first aspect. Therefore, the recombinant collagen exhibits high cell adhesion activity and good hydrophilicity, and can be widely used in cosmetics, medicine, tissue engineering, and other fields.

[0045] According to an embodiment of the present invention, the recombinant collagen has a triple helical conformation, thus having biological activity and can be widely used in various fields such as cosmetology, medicine, and tissue engineering.

[0046] According to an embodiment of the present invention, the recombinant collagen monomer is homologous or heterologous.

[0047] According to an embodiment of the present invention, the recombinant collagen monomers are connected to each other through hydrogen bonds or electrostatic forces.

[0048] In a third aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect.

[0049] In a fourth aspect, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the nucleic acid molecule described in the third aspect.

[0050] In a fifth aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, or expresses the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect.

[0051] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell or a prokaryotic cell.

[0052] According to an embodiment of the present invention, the eukaryotic cell is Pichia pastoris, Saccharomyces cerevisiae, an animal cell or a plant cell.

[0053] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis or Bacillus licheniformis.

[0054] In a sixth aspect, the present invention provides a method for preparing recombinant collagen. According to an embodiment of the present invention, the method comprises: culturing the recombinant cell described in the fifth aspect under conditions suitable for protein expression to obtain the recombinant collagen.

[0055] In the seventh aspect of the present invention, the present invention proposes the use of a reagent in the preparation of a drug, wherein the drug is used to increase the activity of collagen or promote collagen expression or secretion, and the reagent is used to activate collagen active fragments, wherein the collagen active fragments include at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0056] In the eighth aspect of the present invention, the present invention proposes the use of a reagent in the preparation of a drug, wherein the drug is used to inhibit the activity of collagen or inhibit the expression or secretion of collagen, and the reagent is used to inhibit the active fragment of collagen, wherein the active fragment of collagen includes at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0057] According to an embodiment of the present invention, the use of the reagent in preparing a drug may further include at least one of the following additional technical features:

[0058] According to an embodiment of the present invention, the collagen active fragment is derived from amino acids 168 to 1196 of collagen.

[0059] According to an embodiment of the present invention, the collagen active fragment includes three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0060] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 1089 and 1172 of collagen.

[0061] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 234 and 296 of collagen.

[0062] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 273 and 311 of collagen.

[0063] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 288 and 452 of collagen.

[0064] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 300 and 485 of collagen.

[0065] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 444 and 503 of collagen.

[0066] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 483 and 512 of collagen.

[0067] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 501 to 599 of collagen.

[0068] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 507 and 686 of collagen.

[0069] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 591 to 731 of collagen.

[0070] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 678 and 806 of collagen.

[0071] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 726 and 830 of collagen.

[0072] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 795 and 833 of collagen.

[0073] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 798 and 839 of collagen.

[0074] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 852 and 866 of collagen.

[0075] According to an embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 828 and 932 of collagen.

[0076] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 861 and 995 of collagen.

[0077] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 927 and 1094 of collagen.

[0078] According to an embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 987 and 1103 of collagen.

[0079] In the ninth aspect of the present invention, the present invention proposes the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect in the preparation of a medicine for beauty, wound repair, and joint lubrication.

[0080] In the tenth aspect of the present invention, the present invention proposes the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect in preparing food.

[0081] In the eleventh aspect of the present invention, the present invention proposes the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect in beauty, wound repair or joint lubrication.

[0082] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0084] FIG1 is a gel electrophoresis diagram of a recombinant collagen Y109 stock solution according to an embodiment of the present invention;

[0085] FIG2 is a deconvolution diagram of mass spectrometry molecular weight identification of recombinant collagen Y109 according to an embodiment of the present invention;

[0086] FIG3 is a circular dichroism spectrum curve of recombinant collagen Y109 at 20° C. according to an embodiment of the present invention;

[0087] FIG4 is a scatter plot of circular dichroism ellipticity of recombinant collagen Y109 at 221 nm at different temperatures according to an embodiment of the present invention;

[0088] 5 is a protein gel electrophoresis diagram of trypsin-resistant recombinant collagen Y109 according to an embodiment of the present invention (WYM refers to a commercially available similar product);

[0089] FIG6 is a graph showing the test results of the adhesion of different fragments to NIH3T3 cells according to an embodiment of the present invention;

[0090] FIG7 is a graph showing the test results of the adhesion of fragments with different repeat numbers to NIH3T3 cells according to an embodiment of the present invention;

[0091] FIG8 is a graph showing the effects of recombinant collagen Y109 on the adhesion activity of NIH / 3T3 cells within a certain concentration range according to an embodiment of the present invention. DETAILED DESCRIPTION

[0092] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0093] Definition and Description

[0094] Unless otherwise specified or clearly contradicted by context, the articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more." Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., more than one component may be contemplated for use or use in implementing the described embodiments.

[0095] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0096] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0097] The present invention provides a recombinant collagen monomer, recombinant collagen and its use, a nucleic acid molecule, a vector, a recombinant cell and a method for preparing the recombinant collagen, which will be described in detail below.

[0098] Recombinant collagen monomer

[0099] In one aspect of the present invention, a recombinant collagen monomer is provided, wherein the recombinant collagen monomer comprises at least one collagen active fragment, wherein the collagen active fragment comprises at least three identical or different GER sites and / or GEK sites and / or RGD sites and / or GEN sites. Wherein, the "collagen active fragment" described in the present invention refers to a peptide segment from "amino acid 168 to amino acid 1196" of collagen (SEQ ID NO: 1), that is, the GER site, GEK site peptide segment, RGD site, and GEN site peptide segment also come from this interval, wherein multiple GER sites and / or GEK sites and / or RGD sites and / or GEN sites can be continuous or discontinuous.

[0100] It should be noted that the "collagen" referred to in the present invention is native human type III collagen, the full-length amino acid sequence of which is represented by SEQ ID NO: 1. The "amino acids 168 to 1196" herein refer to the amino acid sequence from 168 to 1196 of native human type III collagen, i.e., the helical region of mature human type III collagen.

[0101] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 1089 and 1172 of collagen, corresponding to the sequence shown in SEQ ID NO: 2, i.e., the peptide segment 019 described in the Examples of the present invention. Collagen active fragments within this range have the highest adhesion activity.

[0102] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 234 and 296 of collagen, corresponding to the sequence shown in SEQ ID NO: 7, namely the 001 peptide segment in the examples of the present invention.

[0103] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 273 and 311 of collagen, corresponding to the sequence shown in SEQ ID NO: 8, namely the 002 peptide segment in the examples of the present invention.

[0104] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 288 and 452 of collagen, corresponding to the sequence shown in SEQ ID NO: 9, namely the 003 peptide segment in the examples of the present invention.

[0105] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 300 and 485 of collagen, corresponding to the sequence shown in SEQ ID NO: 10, namely the 004 peptide segment in the examples of the present invention.

[0106] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 444 and 503 of collagen, corresponding to the sequence shown in SEQ ID NO: 11, namely the 005 peptide segment in the examples of the present invention.

[0107] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 483 and 512 of collagen, corresponding to the sequence shown in SEQ ID NO: 12, namely the 006 peptide segment in the examples of the present invention.

[0108] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 501 and 599 of collagen, corresponding to the sequence shown in SEQ ID NO: 13, namely the 007 peptide segment in the examples of the present invention.

[0109] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 507 and 686 of collagen, corresponding to the sequence shown in SEQ ID NO: 14, namely the 008 peptide segment in the examples of the present invention.

[0110] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 591 and 731 of collagen, corresponding to the sequence shown in SEQ ID NO: 15, namely the 009 peptide segment in the examples of the present invention.

[0111] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 678 and 806 of collagen, corresponding to the sequence shown in SEQ ID NO: 16, namely the 010 peptide segment in the examples of the present invention.

[0112] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 726 and 830 of collagen, corresponding to the sequence shown in SEQ ID NO: 17, namely the 011 peptide segment in the examples of the present invention.

[0113] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 795 and 833 of collagen, corresponding to the sequence shown in SEQ ID NO: 18, namely the 012 peptide segment in the examples of the present invention.

[0114] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 798 and 839 of collagen, corresponding to the sequence shown in SEQ ID NO: 19, namely the 013 peptide segment in the examples of the present invention.

[0115] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 852 and 866 of collagen, corresponding to the sequence shown in SEQ ID NO: 20, namely the 014 peptide segment in the examples of the present invention.

[0116] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 828 and 932 of collagen, corresponding to the sequence shown in SEQ ID NO: 21, namely the 015 peptide segment in the examples of the present invention.

[0117] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 861 and 995 of collagen, corresponding to the sequence shown in SEQ ID NO: 22, namely the 016 peptide segment in the examples of the present invention.

[0118] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 927 and 1094 of collagen, corresponding to the sequence shown in SEQ ID NO: 23, namely the 017 peptide segment in the examples of the present invention.

[0119] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 987 and 1103 of collagen, corresponding to the sequence shown in SEQ ID NO: 24, namely the 018 peptide segment in the examples of the present invention.

[0120] In a specific embodiment of the present invention, the collagen active fragment has an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0121] The recombinant collagen monomer of the present invention has no limitation on the number of collagen active fragments, as long as the verification characteristics in the adhesion embodiment can be achieved. Therefore, in the present invention, the recombinant collagen monomer can contain 2 to 12 collagen active fragments, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 collagen active fragments, preferably 6 collagen active fragments. The multiple collagen active fragments contained in the recombinant collagen monomer can be the same or different.

[0122] In a specific embodiment of the present invention, a stabilizing sequence can be added to the C-terminus of at least one collagen active fragment in the recombinant collagen monomer to improve the stability of the collagen active monomer. The amino acid sequence of the stabilizing sequence is shown in SEQ ID NO: 6.

[0123] In a specific embodiment of the present invention, the recombinant collagen monomer has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 25. The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of a protein in which the 019 peptide segment is repeated 6 times in tandem (i.e., the Y109 molecule), and the amino acid sequence shown in SEQ ID NO: 25 is the amino acid sequence of a protein in which the 012 peptide segment is repeated 12 times in tandem (i.e., the Y70 molecule).

[0124] Recombinant collagen

[0125] In another aspect of the present invention, a recombinant collagen is proposed, which is composed of three of the above-mentioned recombinant collagen monomers. The three recombinant collagen monomers can form a triple helix structure, so that the obtained recombinant collagen has high cell adhesion activity, biological activity and good hydrophilicity, and can be widely used in beauty, medicine, tissue engineering and other aspects.

[0126] Nucleic acid molecules, vectors, and recombinant cells

[0127] In yet another aspect, the present invention provides a nucleic acid molecule, vector, and recombinant cell. The nucleic acid molecule encodes the aforementioned recombinant collagen monomer or recombinant collagen protein; the vector comprises the nucleic acid molecule; and the recombinant cell comprises the nucleic acid molecule, the vector, or expresses the aforementioned recombinant collagen monomer or recombinant collagen protein.

[0128] In a specific embodiment of the present invention, the nucleic acid molecule encodes recombinant collagen. The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:4.

[0129] In the above specific embodiments, the "nucleic acid molecule" is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which is a biomacromolecule compound polymerized by many nucleotide monomers and is one of the most basic substances of life. Nucleotide sequence refers to the order of arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA. In some cases, nucleic acid molecules can be modified for use in the vector of the present application, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain a terminal restriction site sequence. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids in one or more given cells or isolated from the one or more given cells.

[0130] In the above-mentioned specific embodiments, the term "vector" refers to a self-replicating DNA molecule used in genetic engineering recombinant DNA technology to transfer DNA segments (target genes) into recipient cells. As the most commonly used and simplest vector in genetic engineering, a vector must consist of three parts: a genetic marker gene, a replication region, and the target gene. In addition to the commonly used E. coli plasmid vector, many other artificially constructed plasmid vectors suitable for use in microorganisms, yeast, plants, and the like have been developed. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules with one or more free ends or no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other polynucleotide types known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, using standard molecular cloning techniques. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell genome upon introduction, thereby replicating along with the host genome. In addition, certain vectors are capable of directing the expression of a gene of interest. Such vectors are referred to herein as "expression vectors." A recombinant expression vector can comprise a format suitable for expressing a nucleic acid in a host cell, meaning that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell used for expression and to which the nucleic acid sequence to be expressed can be operably linked.

[0131] In the above-mentioned specific embodiments, described " host cell " refers to any cell type, which is susceptible to transformation, transfection, transduction, etc. of the nucleic acid construct or expression vector comprising the polynucleotide of the present invention." host cell " covers any offspring of parental cells, which are not identical to the parental cells due to mutations during the replication process. Host cell can be any cell useful in the production of recombinant humanized collagen in the present application. In order to produce recombinant collagen, the nucleic acid encoding the recombinant collagen can be separated and inserted into one or more vectors to further clone and / or express in the host cell. Conventional techniques can be used (for example, by using oligonucleotide probes that can specifically bind to the gene encoding the recombinant collagen) to easily separate and sequence such nucleic acids. Described host cell refers to a cell into which exogenous nucleic acid has been introduced, including the offspring of such cells. Host cell includes transformant and transformed cell, which includes primary transformed cell and offspring derived therefrom, without considering the number of passages. Offspring may not be identical to the parental cell in nucleic acid content, but may contain mutations. Methods for introducing vectors into host cells are well known, such as electroporation. Alternatively, transfection, microinjection, gene gun technology, liposome-mediated delivery, and the like may be used. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli; preferably, the eukaryotic cell is Pichia pastoris.

[0132] Method for preparing recombinant collagen

[0133] In another aspect of the present invention, a method for preparing recombinant collagen is provided, wherein the method comprises culturing the above-mentioned recombinant cells under conditions suitable for protein expression to obtain the recombinant collagen.

[0134] In a specific embodiment of the present invention, the method for preparing recombinant collagen refers to using recombinant DNA or recombinant RNA technology to obtain a recombinant vector connected to a gene segment that can be translated into a target protein, and then transferring the vector into a host cell that can express the target protein to express a specific recombinant protein molecule, thereby obtaining a protein. The culture conditions are well known to those skilled in the art. The present invention does not impose any restrictions on the method of protein expression, which can be confirmed as needed, for example, the method of protein expression is induced expression.

[0135] use

[0136] In another aspect of the present invention, the present invention proposes the use of a reagent in the preparation of a drug, which can be used to increase or inhibit the activity of collagen or the expression or secretion of collagen, and the reagent is used to activate or inhibit the active fragment of collagen, and the active fragment of collagen includes at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0137] In a specific embodiment of the present invention, the amino acid sequence of the GER site, GEK site, and / or RGD site, and / or GEN site in the collagen active fragment is derived from amino acids 168 to 1196 of collagen. The "collagen" described herein is native human type III collagen, the full-length sequence of which is the amino acid sequence set forth in SEQ ID NO: 1. The "amino acids 168 to 1196" herein refers to the amino acid sequence from amino acids 168 to 1196 of native human type III collagen, i.e., the helical region of mature human type III collagen.

[0138] In a preferred embodiment of the present invention, the collagen active fragment is a peptide segment between amino acids 1089 and 1172 of collagen. The collagen active fragment within this range has the highest adhesion activity.

[0139] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 234 and 296 of collagen.

[0140] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 273 and 311 of collagen.

[0141] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 288 and 452 of collagen.

[0142] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 300 and 485 of collagen.

[0143] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 444 and 503 of collagen.

[0144] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 483 and 512 of collagen.

[0145] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 501 and 599 of collagen.

[0146] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 507 and 686 of collagen.

[0147] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 591 and 731 of collagen.

[0148] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 678 and 806 of collagen.

[0149] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 726 and 830 of collagen.

[0150] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 795 and 833 of collagen.

[0151] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 798 and 839 of collagen.

[0152] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 852 and 866 of collagen.

[0153] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 828 and 932 of collagen.

[0154] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 861 and 995 of collagen.

[0155] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 927 and 1094 of collagen.

[0156] In a preferred embodiment of the present invention, the active collagen fragment is a peptide segment between amino acids 987 and 1103 of collagen.

[0157] In another aspect of the present invention, the present invention provides the use of the above-mentioned recombinant collagen monomer or recombinant collagen in the preparation of medicines, which are used for beauty, wound repair, and joint lubrication.

[0158] In another aspect of the present invention, the present invention provides the use of the above-mentioned recombinant collagen monomer or recombinant collagen in preparing food.

[0159] In another aspect of the present invention, the present invention provides the use of the above-mentioned recombinant collagen monomer or recombinant collagen in beauty, wound repair or joint lubrication.

[0160] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0161] Example 1: Selection of protein sequences

[0162] The full-length sequence of natural human type III collagen (Abcam, ab7535) is SEQ ID NO: 1. Based on the amino acid sequences of adhesion-related sites such as GER and GEK, the researchers designed multiple truncated peptides from the helical region of mature human type III collagen (168AA-1196AA), namely peptide 001 (SEQ ID NO: 7), peptide 002 (SEQ ID NO: 8), peptide 003 (SEQ ID NO: 9), peptide 004 (SEQ ID NO: 10), peptide 005 (SEQ ID NO: 11), peptide 006 (SEQ ID NO: 12), peptide 007 (SEQ ID NO: 13), peptide 008 (SEQ ID NO: 14), peptide 009 (SEQ ID NO: 15), peptide 010 (SEQ ID NO: 16), peptide 011 (SEQ ID NO: 17), peptide 012 (SEQ ID NO: 18), peptide 013 (SEQ ID NO: 19), and peptide 014 (SEQ ID NO: 20). NO: 19), peptide 014 (SEQ ID NO: 20), peptide 015 (SEQ ID NO: 21), peptide 016 (SEQ ID NO: 22), peptide 017 (SEQ ID NO: 23), peptide 018 (SEQ ID NO: 24), and peptide 019 (SEQ ID NO: 2). The recombinant protein was expressed in Pichia pastoris and purified. Cell adhesion assays were used to screen for the peptide with the highest adhesion activity, namely peptide 019 (SEQ ID NO: 2), which spans 1089AA-1172AA of the Col3A1 gene (84 amino acids). These 84 amino acids were used as a repeating unit and repeated N times in tandem (N is an integer greater than or equal to 1). No linker was present between the repeats, and a C-terminal stabilizing sequence, namely GPPGPCCGGG (SEQ ID NO: 6), was optionally added to the C-terminus. The present invention does not limit the number of repeats, as long as the adhesion characteristics demonstrated in the examples are achieved. Preferably, the number of repeating sequences is 6, i.e., the Y109 molecule, as shown in SEQ ID NO: 3.

[0163] Example 2: Construction of yeast expression strain

[0164] 1. Targeting the Pichia pastoris expression system, researchers codon-optimized the nucleotide sequence corresponding to the Y109 sequence (SEQ ID NO.3), resulting in the nucleotide sequence SEQ ID NO: 4. They then commissioned BGI to perform gene synthesis, and the synthesized gene fragment was inserted into the pPICZ alpha A expression vector, pPICZ alpha A-Y109, via the XhoI and NotI restriction endonuclease sites. The complete plasmid sequence is SEQ ID NO: 5.

[0165] 2. Linearize the plasmid pPICZ alpha A-Y109 with DraI or SalI, transform it into Pichia pastoris KM71 competent cells by electroporation, and screen the transformants using zeocin resistance plates.

[0166] 3. Small-scale expression identification: Pick 6-8 colonies into 2 mL BMGY medium, culture overnight at 28°C and 220 rpm, and shake until OD 600 = 2 to 6 (logarithmic phase), centrifuge at room temperature, collect the bacterial sludge, replace it with BMMY medium, supplement with 1% methanol every day, and after 3 days of induction expression, use SDS-PAGE to detect the expression of recombinant collagen in the culture medium, and screen the positive clones with higher secretion amount for scale-up production.

[0167] The same method was used to perform small-scale expression tests on 19 short peptide molecules, and the short peptide molecule with the highest expression level was screened. After the secretory expression of the 19 short peptide molecules, the fermentation supernatant was directly concentrated 5 times and then analyzed by SDS-PAGE. The results showed that the expression level of molecule 019 was the highest.

[0168] Example 3: Induced expression of recombinant collagen

[0169] 1. Remove the glycerol seeds from the -80°C ultra-low temperature freezer, re-dissolve at room temperature, and inoculate 0.1% to 5% of the glycerol seeds into the primary seed culture medium (1% yeast extract, 2% tryptone, 2% glycerol and 0.2% histidine). Incubate at 28-30°C, 200-250 rpm for 20-30 h.

[0170] 2. Inoculate into the production tank culture medium (complete BSM inorganic salt basal culture medium) at a ratio of 1-10%, control the growth temperature at 28-30°C, pH = 4-6, and dissolved oxygen greater than or equal to 10%.

[0171] 3. When the dissolved oxygen rises to more than 80%, feed culture medium (including but not limited to glucose, glycerol, yeast powder) is added until the bacterial liquid OD = 200-350, and then feed is stopped.

[0172] 4. After stopping feeding, the dissolved oxygen level returned to 80%. Starve the cells for 0.5-1 hour, cool the cells to 22-28°C, and begin feeding the induction medium at a rate of 2-15 ml / L / hour.

[0173] 5. After 24 to 48 hours of induction, the culture was terminated, the fermentation broth was centrifuged at 10,000 to 12,000 g, and the fermentation supernatant was collected.

[0174] Example 4: Purification process of recombinant collagen

[0175] 1. The supernatant prepared by fermentation in Example 3 was inactivated at 60°C and then separated and purified using an MMC composite chromatography column. The chromatography column was equilibrated with an equilibrium solution (50 mM NaAc, pH 5.0) of not less than 8 column volumes. The fermentation supernatant was loaded, the protein was combined with the chromatography column, washed with a washing solution (20 mM PBS, 1 M NaCl, pH 7.0), and eluted with an eluent (20 mM Tris, 2 M NaCl, pH 9.0), and the eluate was collected.

[0176] 2. Ultrafilter the collected chromatography solution into phosphate solution (20 mM PBS, pH 7.0) and collect the target protein through an anion chromatography column.

[0177] 3. The anion chromatography flow-through is concentrated by ultrafiltration to obtain the collagen stock solution.

[0178] The Y109 collagen stock solution was prepared by the above method, and the prepared Y109 collagen stock solution was subjected to gel electrophoresis detection. The results are shown in Figure 1. The molecular weight is consistent with the theoretical molecular weight, and the purity after preliminary purification is about 85%.

[0179] Example 5: Mass spectrometry detection

[0180] 1. According to Example 4, prepare purified Y109 protein.

[0181] 2. Dilute the Y109 protein with ultrapure water to a final concentration of 1 mg / ml and transfer 200 μL to an injection vial.

[0182] 3. The molecular weight of Y109 was determined by liquid chromatography-mass spectrometry (SEC-ESI-MS). The instrument used was a SCIEX high-resolution mass spectrometry ZenoTOF 7600 system, the chromatographic column was an ACQUITY UPLC Protein BEH SEC, 1 μL was injected, the isocratic elution mode (35% acetonitrile aqueous solution containing 0.1% formic acid) was used, the positive ion mode scan was used, the ion source temperature was set to 450°C, the spray voltage was set to 5500 V, and the primary mass range was set to 300-2000 Da.

[0183] 4. Data processing and analysis were performed using SCIEX Biologics Explorer software. Protein molecular weight identification was based on primary mass spectrometry of intact proteins. Search parameters included no fixed modification, variable modifications for methionine oxidation, glutamine, and asparagine deamination, and the protein library sequence was the Y109 sequence.

[0184] 5. The deconvolution diagram of mass spectrometry molecular weight identification is shown in Figure 2. It can be seen from the figure that the molecular weight after deconvolution is 48784 Da.

[0185] 6. The molecular weights detected by mass spectrometry and the corresponding proteins are shown in Table 1. It can be seen that the detected molecular weight is consistent with the theoretical molecular weight.

[0186] Table 1

[0187] Example 6: Endotoxin Detection

[0188] 1. According to Example 4, prepare purified Y109 protein.

[0189] 2. Prepare reagents, including kinetic colorimetric Limulus amebocyte lysate (Zhanjiang Andus Biological Co., Ltd., 0.005-50 EU / ml), water for bacterial endotoxin test (Xiamen Limulus amebocyte lysate), and a series of working standards for endotoxins used in the photometric method (Zhanjiang Andus Biological Co., Ltd., 0.02 EU / ml, 0.2 EU / ml, and 2 EU / ml).

[0190] 3. Redissolve the photometric working standard endotoxin series into 0.02 EU / ml, 0.2 EU / ml, and 2 EU / ml solutions. Dilute the samples 10, 100, and 2500 times using water suitable for bacterial endotoxin testing.

[0191] 4. After adding the photometric working standard endotoxin series, diluted protein sample, spiked protein sample (positive control), and diluted buffer (negative control) to the microplate in sequence, add the kinetic colorimetric limulus amebocyte lysate reagent reconstituted with bacterial endotoxin test water according to the labeled amount to the microplate containing the above samples.

[0192] 5. Place the microplate into the microplate reader and record the OD value changes within 1 hour at a wavelength of 405 nm and a temperature of 37°C. The corresponding software will display the curve graph synchronously and automatically fit the standard curve to calculate the endotoxin content of the sample.

[0193] After treatment, the Y109 protein can be controlled to 0.6EU / mg.

[0194] Example 7: Detection of recombinant collagen triple helix structure

[0195] To confirm the tertiary structure of recombinant collagen, circular dichroism (CD) was used to analyze collagen. First, recombinant collagen was prepared into a 200 μg / mL protein solution using a 20 mM PB solution. The solution was then allowed to stand overnight at 4°C and subjected to variable-temperature circular dichroism (CD) analysis. The wavelength was set between 190 and 260 nm, the temperature between 4°C and 40°C, and the heating rate was 2°C / min. As shown in the CD spectrum in Figure 3, at low temperatures (20°C), the curve exhibited a negative peak near 195 nm and a positive peak near 221 nm. Furthermore, GraphPad was used to plot the ellipticity at 221 nm at different temperatures, and a fitting curve was drawn. As shown in Figure 4, the positive peak near 221 nm disappeared as the temperature increased, indicating that collagen was a triple helical structure at low temperatures. As the temperature gradually increased, collagen began to unwind until it was completely unwound at 30°C. Based on the curve, the Tm of the Y109 molecule was calculated to be approximately 26°C.

[0196] Example 8: Detection of the triple helix structure of recombinant collagen (anti-Trypsin enzyme cleavage assay)

[0197] Another method for detecting collagen triple helix structure is the trypsin resistance assay. Under specific conditions, a certain concentration of trypsin can degrade single-strand collagen. Once the collagen has formed a triple helix, it resists trypsin cleavage. Therefore, the trypsin resistance assay can be used to analyze collagen triple helix structure. First, recombinant collagen is prepared into a 200 μg / mL protein solution in 20 mM PB solution and allowed to stand overnight at 4°C. Then, 1 ng of trypsin is added per μg of protein, and the protein is digested at different temperatures. After 1 hour of digestion, non-reducing SDS-PAGE loading buffer is added directly and SDS-PAGE is performed. As shown in Figure 5, Y109 is resistant to trypsin cleavage at 4°C and retains a certain degree of triple helical structure at 24°C, indicating that the triple helical structure of Y109 is stable at room temperature.

[0198] Example 9: Cell activity detection

[0199] 1. Screening of fragments with NIH / 3T3 cell adhesion promoting activity

[0200] The candidate fragments (peptide 001 to peptide 019 in Example 1) and controls (commercially available natural human collagen and commercially available recombinant humanized collagen) were diluted with PBS to a consistent final concentration (0.5 mg / ml), and the negative control group (NC) was PBS buffer; 100 μL / well was added to three replicate wells in a 96-well cell culture plate with untreated culture surface; after incubation at 4°C overnight, the cell culture plate was washed once with PBS; a 1% BSA solution was prepared, and 100 μL / well of 1% BSA was added to each well of the above 96-well plate using a spray gun, and incubated at room temperature for 1 hour; after removing the BSA, 200 μL of PBS was added to each well, the wells were washed once, and the PBS was removed; the NIH / 3T3 cell density was adjusted to 1*10 6 cells / mL; inoculated into 96-well plates using a spray gun, 100 μL cells, 1*10 5 cells / well, incubate at 37°C for 1 hour; slowly remove the culture medium and wash four times with PBS; prepare a 96-well plate by adding 10 μL of CCK8 reagent to every 100 μL of culture medium, mix well, add 100 μL / well, incubate at 37°C, 5% CO2 cell culture incubator for 1 hour, then remove; read the absorbance of the 96-well plate at 450 nm with a microplate reader and record the measurement results.

[0201] Natural type III collagen has the activity of promoting NIH / 3T3 cell adhesion. Cells that are not adhered or weakly adhered to the bottom of the well can be removed by washing with PBS. Therefore, the effect of different molecular coatings on cell adhesion activity can be reflected by measuring the number of cells remaining in different wells after PBS washing. After adding CCK8 to the cells, the OD value detected at 450nm is positively correlated with the cell number. 450 The higher the value, the more cells adhered to the well bottom and the better the coated molecule's ability to promote NIH / 3T3 cell adhesion. The experimental results, shown in Figure 6, demonstrate that native human type III collagen indeed exhibits strong NIH / 3T3 cell adhesion activity, with molecule 019 showing particularly pronounced NIH / 3T3 cell adhesion activity among the candidate fragments.

[0202] 2. Y109 promotes the enhancement of NIH3T3 cell adhesion activity

[0203] The inventors further tested the adhesion activity of peptides with varying numbers of tandem repeats in the 019 sequence and found a correlation between adhesion activity and repeat number. Cell adhesion activity was best when the number of repeats was six. Therefore, the molecule with six repeats, Y109 (SEQ ID NO: 3), was selected for further research. The nucleotide sequence encoding Y109 is shown in SEQ ID NO: 4.

[0204] Candidate fragments (019 and Y109) and controls (commercially available natural bovine collagen and commercially available recombinant humanized collagen) were diluted with PBS to a consistent final concentration (0.2 mg / ml). The negative control group (NC) was PBS buffer. 100 μL / well and 3 wells / sample were added to a 96-well cell culture plate with untreated culture surface. After incubation at 4°C overnight, the cell culture plate was washed once with PBS. A 1% BSA solution was prepared and 100 μL / well of 1% BSA was added to each well of the 96-well plate using a dispenser. The plates were incubated at room temperature for 1 hour. After removing the BSA, 200 μL of PBS was added to each well, the wells were washed once, and the PBS was removed. The NIH / 3T3 cell density was adjusted to 1*10 6 cells / mL; inoculated into 96-well plates using a spray gun, 100 μL / well, 1*10 5 Incubate cells / well at 37°C for 1 hour; slowly remove the culture medium and wash four times with PBS (using a plate washer); add 10 μL of CCK8 reagent per 100 μL of culture medium, mix thoroughly, and add to a 96-well plate at 100 μL / well. Incubate at 37°C, 5% CO2 in a cell culture incubator for 1 hour before removing from the plate; read the absorbance of the 96-well plate at 450 nm using a microplate reader and record the measurement results. The experimental results are shown in Figure 7. It can be seen that the Y109 molecule, after six repetitions of the candidate fragment, has stronger adhesion activity to NIH / 3T3 cells than the 019 molecule.

[0205] 3. The Y109 molecule promotes NIH3T3 cell adhesion activity in a concentration-dependent manner

[0206] Y109 and controls (commercially available natural bovine collagen and commercially available recombinant humanized collagen) were diluted to 200 μg / ml with PBS, with a total volume of 255 μL. After mixing, 85 μL was added to 170 μL PBS, and the gradient dilution was performed 7 times in sequence, for a total of 8 concentrations. After the dilution was completed, they were added to a 96-well plate at 50 μL / well, with 3 replicates. The plates were added to a 96-well cell culture plate with untreated culture surfaces. After incubation at 4°C overnight, the cell culture plates were washed once with PBS. A 1% BSA solution was prepared, and 100 μL / well of 1% BSA was added to each well of the 96-well plate using a spray gun, and incubated at room temperature for 1 h. After removing the BSA, 200 μL PBS was added to each well, the wells were washed once, and the PBS was removed. The NIH / 3T3 cell density was adjusted to 1*10 6 cells / mL; inoculated into 96-well plates using a spray gun, 100 μL / well, 1*10 5Incubate cells / well at 37°C for 1 hour; slowly remove the culture medium and wash four times with PBS (using a plate washer). Add 10 μL of CCK8 reagent per 100 μL of culture medium, mix thoroughly, and add 100 μL / well to a 96-well plate. Incubate at 37°C, 5% CO2 in a cell culture incubator for 1 hour before removing from the plate. Read the absorbance of the 96-well plate at 450 nm using a microplate reader and record the measurement results. The experimental results are shown in Figure 8, showing that the Y109 molecule promotes NIH / 3T3 cell adhesion activity within a certain range with increasing concentration.

[0207] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0208] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A recombinant collagen monomer, characterized in that: It comprises at least one active collagen fragment, wherein the active collagen fragment comprises at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

2. The recombinant collagen monomer according to claim 1, characterized in that The collagen active fragment comes from the 168th amino acid to the 1196th amino acid of collagen.

3. The recombinant collagen monomer according to claim 1, characterized in that The collagen active fragment includes three GER sites and / or GEK sites and / or RGD sites and / or GEN sites; The three GER sites and / or GEK sites and / or RGD sites and / or GEN sites are the same or different.

4. The recombinant collagen monomer according to claim 1, characterized in that The collagen active fragment is a peptide segment between the 1089th amino acid and the 1172th amino acid of collagen and / or a peptide segment between the 234th amino acid and the 296th amino acid and / or a peptide segment between the 273rd amino acid and the 311th amino acid and / or a peptide segment between the 288th amino acid and the 452nd amino acid and / or a peptide segment between the 300th amino acid and the 485th amino acid and / or a peptide segment between the 444th amino acid and the 503rd amino acid and / or a peptide segment between the 483rd amino acid and the 512th amino acid and / or a peptide segment between the 501st amino acid and the 599th amino acid and / or a peptide segment between the 507th amino acid and the 686th amino acid and / or a peptide segment between the 597th amino acid and the 686th amino acid. A peptide segment between amino acid 1 and amino acid 731 and / or a peptide segment between amino acid 678 and amino acid 806 and / or a peptide segment between amino acid 726 and amino acid 830 and / or a peptide segment between amino acid 795 and amino acid 833 and / or a peptide segment between amino acid 798 and amino acid 839 and / or a peptide segment between amino acid 852 and amino acid 866 and / or a peptide segment between amino acid 828 and amino acid 932 and / or a peptide segment between amino acid 861 and amino acid 995 and / or a peptide segment between amino acid 927 and amino acid 1094 and / or a peptide segment between amino acid 987 and amino acid 1103.

5. The recombinant collagen monomer according to claim 1, characterized in that: The collagen active fragment has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO:

24.

6. The recombinant collagen monomer according to claim 1, characterized in that: Includes 2 to 12 collagen active fragments; The collagen active fragments are the same or different; The 2 to 12 collagen active fragments are directly or indirectly connected to each other.

7. The recombinant collagen monomer according to claim 6, characterized in that: Includes 6 active collagen fragments.

8. The recombinant collagen monomer according to claim 1, characterized in that Further comprising a stabilizing sequence, wherein the stabilizing sequence is connected to the C-terminus of the at least one collagen active fragment; The amino acid sequence of the stabilizing sequence is SEQ ID NO:

6.

9. The recombinant collagen monomer according to claim 1, characterized in that: The recombinant collagen monomer has an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

25.

10. A recombinant collagen, characterized in that: The invention comprises the recombinant collagen monomers as described in any one of claims 1 to 9.

11. The recombinant collagen according to claim 10, characterized in that The recombinant collagen has a triple helical conformation; The recombinant collagen monomer is homologous or heterologous; The recombinant collagen monomers are connected to each other through hydrogen bonds or electrostatic forces.

12. A nucleic acid molecule, characterized in that Encoding the recombinant collagen monomer according to any one of claims 1 to 9 or the recombinant collagen according to any one of claims 10 to 11.

13. A carrier, characterized in that Comprising the nucleic acid molecule of claim 12.

14. A recombinant cell, characterized in that Comprising the nucleic acid molecule of claim 12, the vector of claim 13 or expressing the recombinant collagen monomer of any one of claims 1 to 9 or the recombinant collagen of any one of claims 10 to 11; The recombinant cell is a eukaryotic cell or a prokaryotic cell; The eukaryotic cell is Pichia pastoris, Saccharomyces cerevisiae, animal cell or plant cell; The prokaryotic cell is Escherichia coli, Bacillus subtilis or Bacillus licheniformis.

15. A method for preparing recombinant collagen, characterized in that: include: The recombinant cell according to claim 14 is cultured under conditions suitable for protein expression to obtain the recombinant collagen.

16. Use of an agent in the preparation of a drug, wherein the drug is used to increase the activity of collagen or promote the expression or secretion of collagen, and the agent is used to activate an active collagen fragment, wherein the active collagen fragment includes at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

17. Use of an agent in the preparation of a drug, wherein the drug is used to inhibit the activity of collagen or inhibit the expression or secretion of collagen, and the agent is used to inhibit the active fragment of collagen, wherein the active fragment of collagen includes at least 3 GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

18. The use according to claim 16 or 17, characterized in that The collagen active fragment comes from the 168th amino acid to the 1196th amino acid of collagen.

19. The use according to claim 16 or 17, characterized in that The collagen active fragment comprises three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

20. The use according to claim 16 or 17, characterized in that The collagen active fragment is a peptide segment between the 1089th amino acid and the 1172th amino acid of collagen and / or a peptide segment between the 234th amino acid and the 296th amino acid and / or a peptide segment between the 273rd amino acid and the 311th amino acid and / or a peptide segment between the 288th amino acid and the 452nd amino acid and / or a peptide segment between the 300th amino acid and the 485th amino acid and / or a peptide segment between the 444th amino acid and the 503rd amino acid and / or a peptide segment between the 483rd amino acid and the 512th amino acid and / or a peptide segment between the 501st amino acid and the 599th amino acid and / or a peptide segment between the 507th amino acid and the 686th amino acid and / or a peptide segment between the 597th amino acid and the 686th amino acid. A peptide segment between amino acid 1 and amino acid 731 and / or a peptide segment between amino acid 678 and amino acid 806 and / or a peptide segment between amino acid 726 and amino acid 830 and / or a peptide segment between amino acid 795 and amino acid 833 and / or a peptide segment between amino acid 798 and amino acid 839 and / or a peptide segment between amino acid 852 and amino acid 866 and / or a peptide segment between amino acid 828 and amino acid 932 and / or a peptide segment between amino acid 861 and amino acid 995 and / or a peptide segment between amino acid 927 and amino acid 1094 and / or a peptide segment between amino acid 987 and amino acid 1103.

21. Use of the recombinant collagen monomer according to any one of claims 1 to 9 or the recombinant collagen according to any one of claims 10 to 11 in the preparation of a drug, wherein the drug is used for beauty, wound repair, and joint lubrication.

22. Use of the recombinant collagen monomer according to any one of claims 1 to 9 or the recombinant collagen according to any one of claims 10 to 11 in preparing food.

23. Use of the recombinant collagen monomer according to any one of claims 1 to 9 or the recombinant collagen according to any one of claims 10 to 11 for beauty, wound repair or joint lubrication.

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