Recombinant humanized type III triple helix collagen, its manufacturing method, and its use

Abstract

This invention proposes the use of recombinant humanized type III triple helix collagen protein and a method for producing the same. Specifically, this invention proposes a recombinant collagen monomer. According to an embodiment of this invention, the recombinant collagen monomer comprises at least one collagen active fragment, the collagen active fragment comprising at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites. The three adhesion sites may be the same or different from each other. The collagen active fragment is derived from amino acids 168 to 1196 of collagen.

Description

Technical Field

[0001] <Priority Information> This application claims the priority and benefit of the patent application with patent application number 2023110945209 filed with the China National Intellectual Property Administration on August 28, 2023, the entire text of which is incorporated herein by reference. The present invention relates to the field of biotechnology, and specifically relates to recombinant humanized type III triple-helix collagen, a method for producing the same and use thereof. Background Art

[0002] Natural collagen (natural collagen protein) is one of the most abundant macromolecular proteins in vivo, accounting for about 20 to 30% of the total protein content. About 28 types of natural collagen have been discovered to date, which are widely distributed in connective tissues of skin, bones, tendons and various organs, and are involved in various life activities such as wound repair, skin aging, blood coagulation, and joint lubrication. As an important biomaterial, collagen has extensive application value in the fields of medicine, cosmetics, food industry and other fields.

[0003] Natural type I and type III collagens are fibrous collagens with complex multilayer structures. They have large molecular weights, and the amino acid sequence of the helical domain in the molecule is a Gly-X-Y repeat sequence, where Y is usually hydroxyproline. Three identical or heterologous protein molecules are arranged through intermolecular interactions such as hydrogen bonding and electrostatic interaction to form a triple-helix structure, and are further arranged in layers to form a higher-order fibrous structure.

[0004] Currently, the main sources of collagen on the market are animal-derived products such as those from pigs, cattle, and fish, and animal-derived collagen is mainly produced by extraction processes using acid hydrolysis, alkaline hydrolysis, or enzymatic hydrolysis. However, animal-derived collagen has the following drawbacks: 1) Because animal-derived collagen has a different amino acid sequence from human-derived collagen, it is immunogenic and carries a risk of allergic reactions. 2) Animal-derived collagen may contain viruses such as mad cow disease prions. 3) The collagen structure may be partially destroyed during the extraction process, potentially reducing its original biological activity. 4) There are religious and ethical issues associated with it.

[0005] Therefore, there is a strong need to develop novel collagen production methods to overcome the above problems. [Overview of the project]

[0006] The present invention aims to solve, to some extent, at least one of the technical problems that exist in the prior art.

[0007] In recent years, with the advancement of synthetic biology, researchers have been using genetic recombination expression technology to produce recombinant human-derived collagen on a large scale, using microorganisms (e.g., E. coli, yeast) as cell factories. Compared to conventional animal-derived collagen extraction processes, the production of recombinant collagen has the following advantages: 1) Recombinant human-derived collagen has the same amino acid sequence as natural human collagen, thus eliminating safety issues caused by animal immunogenicity. 2) Transmission of animal-derived viruses can be avoided. 3) The microbial fermentation process is low-cost, has a short production cycle, and is suitable for large-scale production. In recent years, the Pichia yeast expression system has been widely used for the production of recombinant collagen. The inventors have found that the Pichia yeast expression system combines the advantages of both prokaryotic and eukaryotic expression systems, is easy to operate, offers high expression levels, is low-cost, is suitable for large-scale industrial production, and can impart a certain degree of post-translational modification to foreign proteins. Furthermore, by designing and modifying the Pichia yeast expression system using genetic engineering means, the inventors have found that humanized collagen expressed in yeast has the ability to form a triple helix structure. Humanized collagen, possessing a triple helix structure and biological activity, is a high-order biomaterial similar to natural collagen. In addition, the inventors have discovered that Pichia yeast has high protein secretion efficiency and secretes little of its own protein, allowing for high levels of foreign protein secretion, easy purification, and avoidance of toxic side effects due to the accumulation of fermentation products.

[0008] Accordingly, in a first aspect of the present invention, the present invention provides a recombinant collagen monomer. According to an embodiment of the present invention, the recombinant collagen monomer comprises at least one collagen active fragment, the collagen active fragment comprising at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites. Through continuous experiments, the inventors have found that in natural collagen, related peptide fragments containing at least three adhesion-related sites, namely GER and / or GEK and / or RGD sites and / or GEN sites, are the adhesion-active sites of collagen. Of the peptides obtained by artificially cleaving natural collagen, only peptides containing at least three adhesion-related sites, namely GER and / or GEK and / or RGD sites and / or GEN sites, have adhesion activity or expression or secretion activity in recombinant cells.

[0009] According to embodiments of the present invention, the recombinant collagen monomer further comprises at least one of the following additional technical features. According to embodiments of the present invention, the collagen-active fragment is derived from amino acids 168 to 1196 of collagen.

[0010] According to embodiments of the present invention, the collagen-active fragment comprises three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0011] According to embodiments of the present invention, the three GER sites and / or GEK sites and / or RGD sites and / or GEN sites may be the same or different. For example, they may include three GER sites, or three GEK sites, or two GEK sites and one GER site.

[0012] According to embodiments of the present invention, the collagen active fragment is a peptide located between amino acids 1089 and 1172 of collagen. The inventors have found that the adhesion activity, expression, and secretion levels of the collagen active fragment are highest in this range, thus establishing a basis for the subsequent production of recombinant collagen with high cell adhesion activity.

[0013] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 234th to 296th amino acids of collagen.

[0014] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 273rd to 311th amino acids of collagen.

[0015] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 288th to 452nd amino acids of collagen.

[0016] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 300th to 485th amino acids of collagen.

[0017] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 444th to 503rd amino acids of collagen.

[0018] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 483rd to 512th amino acids of collagen.

[0019] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 501st to 599th amino acids of collagen.

[0020] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 507th to 686th amino acids of collagen.

[0021] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 591st amino acid to the 731st amino acid of collagen.

[0022] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 678th amino acid to the 806th amino acid of collagen.

[0023] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 726th amino acid to the 830th amino acid of collagen.

[0024] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 795th amino acid to the 833rd amino acid of collagen.

[0025] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 798th amino acid to the 839th amino acid of collagen.

[0026] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 852nd amino acid to the 866th amino acid of collagen.

[0027] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 828th amino acid to the 932nd amino acid of collagen.

[0028] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 861st amino acid to the 995th amino acid of collagen.

[0029] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 927th amino acid to the 1094th amino acid of collagen.

[0030] According to an embodiment of the present invention, the collagen active fragment is a peptide between the 987th amino acid to the 1103rd amino acid of collagen.

[0031] According to embodiments of the present invention, the collagen-active fragment is a peptide between amino acids 1089 to 1172 of collagen, and / or a peptide between amino acids 234 to 296, and / or a peptide between amino acids 273 to 311, and / or a peptide between amino acids 288 to 452, and / or a peptide between amino acids 300 to 485, and / or a peptide between amino acids 444 to 503, and / or a peptide between amino acids 483 to 512, and / or a peptide between amino acids 501 to 599, and / or a peptide between amino acids 507 to 686, and / or These are peptides between amino acids 591 and 731, and / or peptides between amino acids 678 and 806, and / or peptides between amino acids 726 and 830, and / or peptides between amino acids 795 and 833, and / or peptides between amino acids 798 and 839, and / or peptides between amino acids 852 and 866, and / or peptides between amino acids 828 and 932, and / or peptides between amino acids 861 and 995, and / or peptides between amino acids 927 and 1094, and / or peptides between amino acids 987 and 1103.

[0032] According to an embodiment of the present invention, 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.

[0033] According to the examples of the present invention, the present invention contains 2 to 12 collagen-active fragments.

[0034] According to the embodiments of the present invention, the present invention contains six collagen-active fragments.

[0035] According to embodiments of the present invention, the collagen-active fragments may be the same or different.

[0036] According to embodiments of the present invention, the 2 to 12 collagen-active fragments are linked together directly or indirectly.

[0037] According to embodiments of the present invention, the invention further includes a stabilizing sequence, the stabilizing sequence being linked to the C-terminus of at least one collagen-active fragment. This improves the stability of the collagen-active monomer.

[0038] According to an embodiment of the present invention, the amino acid sequence of the stabilizing sequence is Sequence ID No. 6.

[0039] According to an embodiment of the present invention, the recombinant collagen monomer has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 25.

[0040] In a second aspect of the present invention, the present invention provides recombinant collagen. According to an embodiment of the present invention, the recombinant collagen contains three recombinant collagen monomers described in the first aspect. As a result, the recombinant collagen has high cell adhesion activity and good hydrophilicity, and can be widely applied in various fields such as cosmetics, medicine, and tissue engineering.

[0041] According to embodiments of the present invention, the recombinant collagen has a triple helix structure. This gives it biological activity, making it widely applicable in various fields such as cosmetics, medicine, and tissue engineering.

[0042] According to the embodiments of the present invention, the recombinant collagen monomers may be of the same origin or of different origins.

[0043] According to embodiments of the present invention, the recombinant collagen monomers are bonded to each other by hydrogen bonds or electrostatic interactions.

[0044] In a third aspect of the present invention, 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.

[0045] In a fourth aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises a nucleic acid molecule as described in the third aspect.

[0046] In a fifth aspect of the present invention, the present invention provides recombinant cells. According to an embodiment of the present invention, the recombinant cells contain a nucleic acid molecule as described in the third aspect, a vector as described in the fourth aspect, or express a recombinant collagen monomer as described in the first aspect or recombinant collagen as described in the second aspect.

[0047] According to embodiments of the present invention, the recombinant cells are eukaryotic cells or prokaryotic cells.

[0048] According to embodiments of the present invention, the eukaryotic cell is a Pichia yeast, a budding yeast, an animal cell, or a plant cell.

[0049] According to embodiments of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, or Bacillus licheniformis.

[0050] In a sixth aspect of the present invention, the present invention provides a method for producing recombinant collagen. According to an embodiment of the present invention, the method includes the step of culturing the recombinant cells described in the fifth aspect under conditions suitable for protein expression, thereby obtaining the recombinant collagen.

[0051] In a seventh aspect of the present invention, the present invention provides the use of a reagent in the manufacture of a pharmaceutical product. The pharmaceutical product is used to improve the activity of collagen or to promote the expression or secretion of collagen, and the reagent is used to activate a collagen active fragment, the collagen active fragment comprising at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0052] In an eighth aspect of the present invention, the present invention provides the use of a reagent in the manufacture of a pharmaceutical product. The pharmaceutical product is used to inhibit the activity of collagen or to inhibit the expression or secretion of collagen, and the reagent is used to inhibit collagen active fragments, the collagen active fragments comprising at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0053] According to embodiments of the present invention, the use of the reagent in the manufacture of pharmaceuticals may further include at least one of the following additional technical features. According to embodiments of the present invention, the collagen-active fragment is derived from amino acids 168 to 1196 of collagen.

[0054] According to embodiments of the present invention, the collagen-active fragment comprises three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0055] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 1089th to 1172nd amino acids of collagen.

[0056] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 234th to 296th amino acids of collagen.

[0057] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 273rd to 311th amino acids of collagen.

[0058] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 288th to 452nd amino acids of collagen.

[0059] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 300th to 485th amino acids of collagen.

[0060] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 444th to 503rd amino acids of collagen.

[0061] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 483rd to 512th amino acids of collagen.

[0062] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 501st to 599th amino acids of collagen.

[0063] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 507th to 686th amino acids of collagen.

[0064] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 591st to 731st amino acids of collagen.

[0065] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 678th to 806th amino acids of collagen.

[0066] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 726th and 830th amino acids of collagen.

[0067] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 795th to 833rd amino acids of collagen.

[0068] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 798th to 839th amino acids of collagen.

[0069] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 852nd to 866th amino acids of collagen.

[0070] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 828th to 932nd amino acids of collagen.

[0071] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 861st to 995th amino acids of collagen.

[0072] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 927th to 1094th amino acids of collagen.

[0073] According to an embodiment of the present invention, the collagen-active fragment is a peptide located between the 987th to 1103rd amino acids of collagen.

[0074] In a ninth aspect of the present invention, the present invention provides the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect in the manufacture of pharmaceuticals. The pharmaceuticals are used for cosmetic purposes, wound healing, and joint lubrication.

[0075] In a tenth aspect of the present invention, the present invention provides the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect in food manufacturing.

[0076] In an eleventh aspect of the present invention, the present invention provides the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect for cosmetic purposes, wound repair, or joint lubrication.

[0077] Additional aspects and advantages of the present invention are described in part in the following description, will become apparent from the following description, or will be understood through the practice of the present invention. [Brief explanation of the drawing]

[0078] The above and / or additional aspects and advantages of the present invention will become apparent and readily understandable from the description of the embodiments with reference to the following drawings. [Figure 1] This is a gel electrophoresis diagram of the recombinant collagen Y109 stock solution according to an example of the present invention. [Figure 2] This is a deconvolution diagram showing the molecular weight identification of recombinant collagen Y109 by mass spectrometry according to an embodiment of the present invention. [Figure 3] This is a circular dichroism spectral curve diagram of recombinant collagen Y109 according to an embodiment of the present invention at 20°C. [Figure 4] This is a scatter plot of the circular dichroism ellipticity at 221 nm under different temperature conditions for recombinant collagen Y109 according to an embodiment of the present invention. [Figure 5] This is a protein gel electrophoresis diagram showing the trypsin-resistant enzyme digestion of recombinant collagen Y109 according to an embodiment of the present invention (where WYM indicates a commercially available similar product). [Figure 6] This figure shows the test results of the effect of different fragments according to the embodiment of the present invention on NIH3T3 cell adhesion. [Figure 7] This figure shows the test results of the effect of different repeat number fragments according to embodiments of the present invention on NIH3T3 cell adhesion. [Figure 8] This figure shows the results of the effect of recombinant collagen Y109 in a certain concentration range on NIH / 3T3 cell adhesion activity according to an example of the present invention. [Modes for carrying out the invention]

[0079] The following describes in detail embodiments of the present invention. The embodiments described below are illustrative and are for illustrative purposes only, and should not be construed as limiting the present invention.

[0080] <Definition and Explanation> Unless otherwise stated or unless there is a clear contextual inconsistency, the articles “one,” “one (kind),” and “the foregoing” 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 (i.e., at least one) subject. For example, “one component” refers to one or more components, meaning that multiple components may be employed or used in the embodiment.

[0081] In this specification, the terms "includes" or "contains" are open expressions and include the content explicitly stated in the present invention, but do not exclude other content.

[0082] In this specification, the terms “optional,” “any,” or “optional” mean that the events or conditions described below may or may not occur, and include both cases in which such events or conditions occur and cases in which they do not occur.

[0083] This invention provides recombinant collagen monomers, recombinant collagen and its uses, nucleic acid molecules, vectors, recombinant cells, and methods for producing recombinant collagen, each of which will be described in detail below.

[0084] <Recombinant collagen monomers> In one aspect of the present invention, the present invention provides a recombinant collagen monomer. The recombinant collagen monomer comprises at least one collagen active fragment, the collagen active fragment comprising at least three identical or different GER sites and / or GEK sites and / or RGD sites and / or GEN sites. Here, "collagen active fragment" in the present invention refers to a peptide derived from amino acids between position 168 and 1196 of collagen (SEQ ID NO: 1), that is, the GER sites, GEK sites, RGD sites and GEN sites are also peptides derived from this section. Here, the multiple GER sites and / or GEK sites and / or RGD sites and / or GEN sites may be consecutive or discontinuous.

[0085] In this invention, "collagen" refers to natural human type III collagen, and its full-length sequence is the amino acid sequence shown in Sequence ID No. 1. Furthermore, "amino acid position 168 to amino acid position 1196" refers to the amino acid sequence from position 168 to position 1196 of natural human type III collagen, i.e., the helical region of mature human type III collagen.

[0086]

[0087] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 1089 and 1172 of collagen, corresponding to the sequence shown in SEQ ID NO: 2. That is, the 019 peptide in the examples of the present invention. Collagen-active fragments within this range exhibit the highest adhesion activity.

[0088] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 234 and 296 of collagen, corresponding to the sequence shown in Sequence ID No. 7. That is, it is the 001 peptide in the examples of the present invention.

[0089] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 273 to 311 of collagen, corresponding to the sequence shown in SEQ ID NO: 8. That is, it is the 002 peptide in the examples of the present invention.

[0090] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 288 and 452 of collagen, corresponding to the sequence shown in SEQ ID NO: 9. That is, it is the 003 peptide in the example of the present invention.

[0091] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 300th to 485th amino acids of collagen, corresponding to the sequence shown in SEQ ID NO: 10. That is, it is the 004 peptide in the examples of the present invention.

[0092] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 444 and 503 of collagen, corresponding to the sequence shown in SEQ ID NO: 11. That is, it is the 005 peptide in the examples of the present invention.

[0093] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 483 to 512 of collagen, corresponding to the sequence shown in SEQ ID NO: 12. That is, it is the 006 peptide in the examples of the present invention.

[0094] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 501st to 599th amino acids of collagen, corresponding to the sequence shown in SEQ ID NO: 13. That is, it is the 007 peptide in the examples of the present invention.

[0095] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 507 and 686 of collagen, corresponding to the sequence shown in SEQ ID NO: 14. That is, it is the 008 peptide in the examples of the present invention.

[0096] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 591 to 731 of collagen, corresponding to the sequence shown in SEQ ID NO: 15. That is, it is the 009 peptide in the examples of the present invention.

[0097] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 678 and 806 of collagen, corresponding to the sequence shown in SEQ ID NO: 16. That is, it is the 010 peptide in the examples of the present invention.

[0098] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 726 and 830 of collagen, corresponding to the sequence shown in SEQ ID NO: 17. That is, it is the 011 peptide in the examples of the present invention.

[0099] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 795 and 833 of collagen, corresponding to the sequence shown in SEQ ID NO: 18. That is, it is the 012 peptide in the examples of the present invention.

[0100] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 798 and 839 of collagen, corresponding to the sequence shown in SEQ ID NO: 19. That is, it is the 013 peptide in the examples of the present invention.

[0101] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 852 to 866 of collagen, corresponding to the sequence shown in SEQ ID NO: 20. That is, it is the 014 peptide in the examples of the present invention.

[0102] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 828 and 932 of collagen, corresponding to the sequence shown in SEQ ID NO: 21. That is, it is the 015 peptide in the examples of the present invention.

[0103] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 861 to 995 of collagen, corresponding to the sequence shown in SEQ ID NO: 22. That is, it is the 016 peptide in the examples of the present invention.

[0104] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 927 and 1094 of collagen, corresponding to the sequence shown in SEQ ID NO: 23. That is, it is the 017 peptide in the examples of the present invention.

[0105] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 987 to 1103 of collagen, corresponding to the sequence shown in SEQ ID NO: 24. That is, it is the 018 peptide in the examples of the present invention.

[0106] In one specific embodiment of the present invention, 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.

[0107] GPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGER(Sequence ID 2) GRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAP(Sequence ID 7) GEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRP(Sequence No. 8) GLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEA (Sequence ID 9) GPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGER(Sequence ID 10) GPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEK(Sequence ID 11) GERGAPGFRGPAGPNGIPGEKGPAGERGAP(Sequence ID 12) GEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGP(Sequence ID 13) GERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPP(Sequence ID 14) GKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGER (Sequence ID 15) GAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPP (Sequence ID 16) GMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAP(Sequence ID 17) GSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEK (Sequence ID 18) GERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPP(Sequence ID 19) GERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSP (Sequence ID 20) GAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEK (Sequence ID 21) GERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPP(Sequence ID 22) GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDK (Sequence ID 23) GLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAA (Sequence ID 24)

[0108] There are no particular restrictions on the number of collagen active fragments in the recombinant collagen monomer of the present invention; it is arbitrary as long as the adhesive properties verified in the examples can be achieved. Therefore, in the present invention, the recombinant collagen monomer may contain 2 to 12 collagen active fragments, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 collagen active fragments. Preferably, it contains 6 collagen active fragments. Here, the multiple collagen active fragments contained in the recombinant collagen monomer may be the same or different.

[0109] In one specific embodiment of the present invention, a stabilizing sequence may be added to the C-terminus of at least one collagen active fragment in the recombinant collagen monomer, thereby improving the stability of the collagen active monomer. Here, the amino acid sequence of the stabilizing sequence is shown in SEQ ID NO: 6.

[0110] GPPGPCCGGG(Sequence ID 6)

[0111] In one specific embodiment of the present invention, the recombinant collagen monomer has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 25. Here, the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of a protein in which the 019 peptide is repeated in series six times (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 is repeated in series twelve times (i.e., the Y70 molecule).

[0112] GPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGEGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPG PAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGERGPRGDKGE TGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGPPGPCCGGG(Sequence ID 3) GSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPG ERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGET GPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGPPGPCCGGG(Sequence ID 25)

[0113] <Recombinant Collagen> In another aspect of the present invention, the present invention provides recombinant collagen. The recombinant collagen is composed of three recombinant collagen monomers, and these three recombinant collagen monomers can form a triple helix structure. As a result, the resulting recombinant collagen has high cell adhesion activity, biological activity and good hydrophilicity, and can be widely applied in various fields such as cosmetics, medicine, and tissue engineering.

[0114] <Nucleic acid molecules, vectors, and recombinant cells> In yet another aspect of the present invention, the present invention provides a nucleic acid molecule, a vector, and a recombinant cell, wherein the nucleic acid molecule is used to encode the recombinant collagen monomer or recombinant collagen, the vector comprises the nucleic acid molecule, and the recombinant cell comprises the nucleic acid molecule and the vector, or expresses the recombinant collagen monomer or recombinant collagen.

[0115] In one specific embodiment of the present invention, the nucleic acid molecule encodes recombinant collagen. The nucleotide sequence of the nucleic acid molecule is shown in Sequence ID No. 4.

[0116]

[0117] In the specific embodiments described above, the term "nucleic acid molecule" refers to a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biomacromolecules formed by the polymerization of numerous nucleotide monomers and are one of the most fundamental substances constituting life. A nucleotide sequence refers to the sequence of bases in DNA or RNA. The nucleic acid molecule includes cDNA, and in some cases, the nucleic acid molecule may be modified for use in the vector of this application, for example, for the purpose of codon optimization. Alternatively, the sequence may be designed to include terminal restriction enzyme sequences for the purpose of cloning into a vector. Nucleic acid molecules can be obtained from various sources, for example, by amplifying coding nucleic acids present in or isolated from one or more given cells by polymerase chain reaction (PCR).

[0118] In the specific embodiments described above, the term "vector" refers to a self-replicating DNA molecule used in genetic engineering recombinant DNA technology to transfer a DNA fragment (target gene) to a recipient cell. The most common and convenient vector in genetic engineering must contain at least three elements: a selection marker gene, a replication region, and the target gene. In addition to E. coli plasmid vectors, a variety of artificially constructed plasmid vectors applicable to microorganisms, yeast, plants, etc., have also been developed. Vectors, though not limited to these, include: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules with or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA fragments can be inserted by standard molecular cloning techniques. Some vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors), while other vectors (e.g., non-episomal mammalian vectors) are integrated into the host genome after introduction into the host cell and replicate together with the host genome. Furthermore, some vectors can be directed to the expression of a target gene, and such vectors are referred to herein as “expression vectors.” Recombinant expression vectors include a form suitable for expressing nucleic acids in host cells, that is, they include one or more regulatory elements selected based on the host cell used for expression and operably linked to the nucleic acid sequence to be expressed.

[0119] In the specific embodiments described above, the term "host cell" refers to any cell type that is readily subjected to transformation, transfection, or transduction by a nucleic acid construct or expression vector containing the polynucleotide of the present invention. The "host cell" includes all offspring of the parent cell that are not entirely identical to the parent cell due to mutations during replication. The host cell can be any cell useful for the production of recombinant humanized collagen of the present application. To produce recombinant collagen, the nucleic acid encoding recombinant collagen can be isolated, inserted into one or more vectors, and cloned and / or expressed in host cells. Such nucleic acids can be easily isolated and sequenced, for example, by using oligonucleotide probes that can specifically bind to genes encoding recombinant collagen. Host cells refer to cells into which foreign nucleic acids have been introduced, and this includes the offspring of those cells. Host cells include transformants and transformed cells, including primary transformed cells and their descendants, regardless of the number of passages. The offspring may not be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Methods for introducing vectors into host cells are well known and include, for example, electroporation, transfection, microinjection, gene gun method, and liposome-mediated method. The host cell is either a prokaryotic cell or a eukaryotic cell, selected from Pichia yeast, budding yeast, Escherichia coli, or Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli, and preferably, the eukaryotic cell is Pasteur Pichia yeast (Pichia pastoris).

[0120] <Method for producing recombinant collagen> In yet another aspect of the present invention, the present invention provides a method for producing recombinant collagen. The method comprises culturing the recombinant cells under conditions suitable for protein expression, thereby obtaining the recombinant collagen.

[0121] In one specific embodiment of the present invention, a method for producing recombinant collagen refers to a method of obtaining a protein by using recombinant DNA or recombinant RNA technology to obtain a recombinant vector to which gene fragments translatable to a target protein are linked, and then introducing this vector into a host cell capable of expressing the target protein to express a specific recombinant protein molecule. The culture conditions are known to those skilled in the art, and the present invention does not particularly limit the protein expression method, which may be confirmed as necessary; for example, it may be induced expression.

[0122] <Use> In yet another aspect of the present invention, the present invention provides the use of a reagent in the manufacture of a pharmaceutical product. The pharmaceutical product is used to promote or inhibit the activity of collagen or the expression or secretion of collagen, and the reagent is used to activate or inhibit collagen active fragments. The collagen active fragments include at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

[0123] In one specific embodiment of the present invention, the amino acid sequences of the GER site, GEK site and / or RGD site and / or GEN site in the collagen active fragment are derived from amino acids 168 to 1196 of collagen. Here, "collagen" refers to natural human type III collagen, and its full-length sequence is the amino acid sequence shown in Sequence ID No. 1. Furthermore, "amino acids 168 to 1196" refers to the amino acid sequence 168 to 1196 of natural human type III collagen, i.e., the helical region of mature human type III collagen.

[0124] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between amino acids 1089 and 1172 of collagen. Collagen-active fragments within this range exhibit the highest adhesion activity.

[0125] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 234th to 296th amino acids of collagen.

[0126] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 273rd to 311th amino acids of collagen.

[0127] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 288th to 452nd amino acids of collagen.

[0128] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 300th to 485th amino acids of collagen.

[0129] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 444th to 503rd amino acids of collagen.

[0130] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 483rd to 512th amino acids of collagen.

[0131] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 501st to 599th amino acids of collagen.

[0132] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 507th to 686th amino acids of collagen.

[0133] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 591st to 731st amino acids of collagen.

[0134] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 678th to 806th amino acids of collagen.

[0135] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 726th to 830th amino acids of collagen.

[0136] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 795th to 833rd amino acids of collagen.

[0137] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 798th to 839th amino acids of collagen.

[0138] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 852nd to 866th amino acids of collagen.

[0139] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 828th to 932nd amino acids of collagen.

[0140] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 861st to 995th amino acids of collagen.

[0141] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 927th to 1094th amino acids of collagen.

[0142] In one preferred embodiment of the present invention, the collagen-active fragment is a peptide located between the 987th to 1103rd amino acids of collagen.

[0143] In yet another aspect of the present invention, the present invention provides the use of the above-mentioned recombinant collagen monomer or recombinant collagen in the manufacture of a pharmaceutical product, said pharmaceutical product used for cosmetic purposes, wound healing, and joint lubrication.

[0144] In yet another aspect of the present invention, the present invention provides the use of the above-mentioned recombinant collagen monomer or recombinant collagen in the production of food products.

[0145] In yet another aspect of the present invention, the present invention provides the use of the above-mentioned recombinant collagen monomer or recombinant collagen for cosmetic purposes, wound repair, or joint lubrication.

[0146] The following describes examples of the present invention in detail. The examples described below are illustrative and are for illustrative purposes only, and should not be construed as limiting the present invention. Where specific technical terms or conditions are not explicitly stated in the examples, the technical terms or conditions described in the relevant art literature or the product's instruction manual shall be followed. Unless the manufacturer is explicitly stated for the reagents or equipment used, they are all commonly available commercial products.

[0147] Example 1: Selection of protein sequence The full-length sequence of natural human type III collagen (Abcam, ab7535) is sequence number 1. Based on the amino acid sequences of adhesion-related sites such as GER and GEK, researchers designed multiple cleavage peptides from the helical region (168AA~1196AA) of mature human type III collagen. Specifically, these are peptide 001 (sequence number 7), peptide 002 (sequence number 8), peptide 003 (sequence number 9), peptide 004 (sequence number 10), peptide 005 (sequence number 11), peptide 006 (sequence number 12), peptide 007 (sequence number 13), peptide 008 (sequence number 14), peptide 009 (sequence number 15), peptide 010 (sequence number 16), peptide 011 (sequence number 17), peptide 012 (sequence number 18), peptide 013 (sequence number 19), peptide 014 (sequence number 20), peptide 015 (sequence number 21), peptide 016 (sequence number 22), peptide 017 (sequence number 23), peptide 018 (sequence number 24), and peptide 019 (sequence number 2). Recombinant proteins were expressed using Pichia yeast, and after purification, the fragment with the highest adhesion activity was selected by cell adhesion testing. As a result, the 019 peptide (SEQ ID NO: 2), which corresponds to 1089AA~1172AA of the Col3A1 gene (84 amino acids), was obtained. These 84 amino acids were used as a repeat unit and repeated in series N times (N is an integer of 1 or more), without linkers between the repeat sequences. If necessary, a C-terminal stabilization sequence, i.e., GPPGPCCGGG (SEQ ID NO: 6), may be added to the C-terminus. The present invention does not particularly limit the number of repeat sequences, but it is required that the adhesion properties verified in the examples be achieved. Preferably, the number of repeat sequences is 6, i.e., the Y109 molecule, as shown in SEQ ID NO: 3.

[0148] Example 2: Construction of a yeast expression strain 1. For the Pichia yeast expression system, the researchers performed codon optimization on the nucleotide sequence corresponding to the Y109 sequence (SEQ ID NO: 3), and designated this nucleotide sequence as SEQ ID NO: 4. Gene synthesis was commissioned to Hua Da Genetics, and the resulting gene fragment was inserted into the pPICZ alpha A expression vector via the XhoI and NotI restriction enzyme sites to construct pPICZ alpha A-Y109. The complete sequence of this plasmid is shown as SEQ ID NO: 5.

[0149]

[0150] 2. Plasmid pPICZ alpha A-Y109 was linearized and digested using DraI or SalI, then introduced into competent Pichia yeast KM71 cells by electroporation, and transformants were selected using zeocin-resistant plates.

[0151] 3. Small-scale expression evaluation: 6-8 colonies were inoculated into 2 mL of BMGY medium and cultured overnight at 28°C and 220 rpm. After shaking, the OD600 reached 2-6 (logarithmic growth phase), then the cells were collected by centrifugation at room temperature and replaced with BMMY medium. Induced expression was induced for 3 days by supplementing with 1% methanol daily, and recombinant collagen expression in the medium was detected by SDS-PAGE. Positive clones with high secretion levels were selected and used for scale-up production.

[0152] For the 19 short peptide molecules, small-scale expression evaluation was performed using the same method as described above, and the short peptide molecule with the highest expression level was selected. After secretory expression of the 19 short peptide molecules, the fermentation supernatant was directly concentrated 5-fold and analyzed by SDS-PAGE, confirming that molecule O19 had a high expression level.

[0153] Example 3: Inducible expression of recombinant collagen 1. Glycerol seeds were removed from an ultra-low temperature freezer at -80°C, restored at room temperature, and then inoculated at a concentration of 0.1% to 5% in primary seed medium (1% yeast extract, 2% tryptone, 2% glycerol, 0.2% histidine). The cultures were then incubated at 28-30°C and 200-250 rpm for 20-30 hours.

[0154] 2.1-10% were inoculated into the culture medium in the production tank (complete BSM inorganic salt base medium), and the growth temperature was controlled to 28-30°C, pH = 4-6, and dissolved oxygen level above 10%.

[0155] 3. After the dissolved oxygen level recovered to over 80%, feed-batch supplementation medium (containing, but not limited to, glucose, glycerol, yeast powder, etc.) was added, and supplementation was stopped when the OD of the bacterial suspension reached 200-350.

[0156] 4. After stopping the supply of oxygen, once the dissolved oxygen level recovered to 80%, the subjects were kept in a starvation state for 0.5 to 1 hour, the temperature was lowered to 22 to 28°C, and induction expression was initiated by feeding the induction medium. The flow rate was set to 2 to 15 mL / L·h.

[0157] 5. After induction for 24 to 48 hours, the culture was terminated, and the fermentation liquid was centrifuged with a centrifugal force of 10,000 to 12,000 g to collect the fermentation supernatant.

[0158] Example 4: Purification process of recombinant collagen 1. The fermentation supernatant obtained in Example 3 was inactivated at high temperature at 60°C and then separated and purified using an MMC composite chromatography column. The column was equilibrated with an equilibrium solution (50 mM NaAc, pH 5.0) of 8 column volumes or more, and the fermentation supernatant was loaded. After binding the protein to the column, it was washed with a washing solution (20 mM PBS, 1 M NaCl, pH 7.0), eluted with an eluent (20 mM Tris, 2 M NaCl, pH 9.0), and the eluted fraction was collected.

[0159] 2. The chromatographic recovery solution was replaced with phosphate buffer (20 mM PBS, pH 7.0) by ultrafiltration, and the target protein was recovered by passing it through an anion exchange chromatography column.

[0160] 3. The phosphate obtained from anion exchange chromatography was concentrated by ultrafiltration to obtain the collagen stock solution.

[0161] Figure 1 shows the results of gel electrophoresis performed on the Y109 collagen stock solution prepared using the method described above. The molecular weight was consistent with the theoretical molecular weight, and the purity after primary purification was approximately 85%.

[0162] Example 5: Mass Spectrometry Measurement 1. Purified Y109 protein was prepared according to Example 4.

[0163] 2. The Y109 protein was diluted with ultrapure water to a final concentration of 1 mg / mL, and 200 μL was dispensed into a vial.

[0164] 3. The molecular weight of Y109 was measured by liquid chromatography-mass spectrometry (SEC-ESI-MS). The instrument used was a SCIEX ZenoTOF 7600 high-resolution mass spectrometer, with an ACQUITY UPLC Protein BEH SEC column, an injection volume of 1 μL, and isocratic elution mode (35% acetonitrile aqueous solution containing 0.1% formic acid). Scanning was performed in positive ion mode. The ion source temperature was set to 450°C, the spray voltage to 5500V, and the primary mass range to 300-2000 Da.

[0165] 4. Data analysis was performed using SCIEX Biologics Explorer software. Protein molecular weight identification was performed based on the primary mass spectrum of the full-length protein. The search criteria were no fixed modification, methionine oxidation, and deamidation of glutamine and asparagine as variable modifications. The protein database sequence used was the Y109 sequence.

[0166] 5. The deconvolution diagram for molecular weight identification by mass spectrometry is shown in Figure 2. From the figure, it can be seen that the molecular weight after deconvolution is 48784 Da.

[0167] 6. The detected molecular weights and corresponding proteins are shown in Table 1. The detected molecular weights were confirmed to be consistent with the theoretical molecular weights.

[0168] [Table 1]

[0169] Example 6: Endotoxin test 1. Purified Y109 protein was prepared according to Example 4.

[0170] 2. Reagents were prepared. These included Limulus reagent for dynamic colorimetric testing (Zhanjiang Andus Biotechnology Co., Ltd., 0.005~50 EU / mL), water for bacterial endotoxin testing (Xiamen Limulus reagent), and a series of standard endotoxins specifically for colorimetric testing (Zhanjiang Andus Biotechnology, 0.02 EU / mL, 0.2 EU / mL, 2 EU / mL).

[0171] 3. The standard endotoxin series for colorimetric testing was redissolved to prepare solutions of 0.02 EU / mL, 0.2 EU / mL, and 2 EU / mL. The samples were diluted 10-fold, 100-fold, and 2500-fold using bacterial endotoxin testing water.

[0172] 4. The standard endotoxin series for colorimetric methods, diluted protein samples, protein samples with added spikes (positive control), and diluted buffer (negative control) were sequentially added to a microplate. Then, Limulus reagent for dynamic colorimetric methods, redissolved in bacterial endotoxin testing water according to the indicated amount, was added to the microplate containing the samples.

[0173] 5. The microplate was placed in a microplate reader, and the OD value change within one hour was recorded under conditions of a wavelength of 405 nm and a temperature of 37°C. The curve was displayed simultaneously using the corresponding software, and the endotoxin content in the sample was calculated by automatic fitting of the standard curve.

[0174] The endotoxin content in the Y109 protein after treatment could be controlled to 0.6 EU / mg.

[0175] Example 7: Detection of the triple helix structure of recombinant collagen To confirm the tertiary structure of recombinant collagen, the collagen protein was measured by circular dichroism (CD) spectroscopy. First, recombinant collagen was prepared as a 200 μg / mL protein solution using 20 mM PB solution, and after standing overnight at 4°C, temperature-variable CD measurements were performed. The wavelength was set to 190-260 nm, the temperature to 4°C-40°C, and the heating rate to 2°C / min. As shown in the CD spectrum (Figure 3), at low temperatures (20°C), a negative peak was observed around 195 nm and a positive peak around 221 nm. Furthermore, using GraphPad, the ellipticity at 221 nm at different temperatures was plotted as a scatter plot, and a fitting curve was created (Figure 4). Since the positive peak around 221 nm disappeared with increasing temperature, it was shown that collagen has a triple helix structure at low temperatures, and dehelixification progresses with increasing temperature, with complete dehelixification occurring at 30°C. The Tm of the Y109 molecule, calculated based on the curve, was approximately 26°C.

[0176] Example 8: Detection of recombinant collagen triple helix structure (trypsin resistance test) Another method for detecting the collagen triple helix structure was performed using a trypsin resistance test. Under specific conditions, a certain concentration of trypsin degrades single-chain collagen molecules, but once the collagen molecule forms a triple helix structure, it becomes resistant to trypsin digestion. Therefore, this test allows for the analysis of the triple helix structure. First, recombinant collagen was prepared as a 200 μg / mL protein solution using 20 mM PB solution and allowed to stand overnight at 4°C. Enzyme digestion was performed at a ratio of 1 ng of trypsin per 1 μg of protein under different temperature conditions. After 1 hour of digestion, a non-reducing SDS-PAGE sample buffer was directly added, and SDS-PAGE was performed. As shown in Figure 5, the Y109 molecule showed resistance to trypsin digestion at 4°C and maintained a certain degree of triple helix structure even at 24°C. Therefore, it was confirmed that the triple helix structure of the Y109 molecule is stable at room temperature.

[0177] Example 9: Evaluation of cell activity 1. Screening for fragments that promote NIH / 3T3 cell adhesion activity Candidate fragments (peptides 001-019 from Example 1) and controls (commercially available natural human collagen, commercially available recombinant humanized collagen) were diluted to a final concentration of 0.5 mg / mL using PBS, and PBS buffer was used as a negative control (NC). 100 μL / well, 3 replicates were added to an untreated 96-well cell culture plate. After incubation overnight at 4°C, the plates were washed once with PBS. A 1% BSA solution was prepared, and 100 μL / well was added to each well using a multichannel pipette, and incubated at room temperature for 1 hour. After BSA removal, each well was washed once with 200 μL of PBS, and the PBS was removed. The NIH / 3T3 cell density was set to 1 × 10⁶ 6 Prepare the solution to the required number of cells / mL and fill a 96-well plate with 100 μL / well (1 × 10⁶ 5 Cells were seeded (per well) and incubated at 37°C for 1 hour. The medium was carefully removed and washed four times with PBS. The CCK8 reagent was prepared at a ratio of 10 μL per 100 μL of medium, mixed, and then added to a 96-well plate at a rate of 100 μL / well. The cells were incubated in a cell incubator at 37°C under 5% CO2 conditions for 1 hour. The absorbance at 450 nm was measured using a microplate reader and the results were recorded.

[0178] Natural type III collagen has activity that promotes the adhesion of NIH / 3T3 cells. Since PBS washing can remove unadhered or weakly adhered cells from the bottom of the well, the effect of each molecular coating on cell adhesion activity can be evaluated by measuring the number of cells remaining after PBS washing. After adding CCK8, the OD value measured at 450 nm showed a positive correlation with the number of cells, and a higher OD450 indicated a larger number of cells adhered to the bottom of the well and a higher NIH / 3T3 cell adhesion promoting activity of the molecule. From the experimental results (Figure 6), natural human type III collagen has extremely high NIH / 3T3 cell adhesion promoting activity, and among the candidate fragments, molecule 019 showed remarkable adhesion promoting activity.

[0179] 2. Enhancement of NIH / 3T3 cell adhesion-promoting activity by Y109 The inventors evaluated the adhesion activity of molecules with different numbers of serial repeats based on the O19 peptide sequence and found that adhesion activity correlated with the number of repeats. Since cell adhesion activity was highest with 6 repeats, the Y109 molecule (SEQ ID NO: 3) with 6 repeats was selected for subsequent research. The nucleotide sequence encoding the Y109 molecule is SEQ ID NO: 4.

[0180] Candidate fragments (019 and Y109) and controls (commercially available natural bovine collagen and commercially available recombinant humanized collagen) were diluted to the same final concentration (0.2 mg / mL) using PBS, and PBS buffer was used as a negative control (NC). Each sample was added to a 96-well cell culture plate with an untreated culture surface at a rate of 100 μL / well, with 3 wells per sample. After incubation overnight at 4°C, the cell culture plate was washed once with PBS. Next, a 1% BSA solution was prepared and added to each well of the 96-well cell culture plate using a multichannel pipette at a rate of 100 μL / well, and incubated at room temperature for 1 hour. After removing the BSA solution, each well was washed once with 200 μL of PBS, and the PBS was removed. The cell density of NIH / 3T3 cells was set to 1 × 10⁶. 6 Prepare the solution to cells / mL and use a multichannel pipette to transfer 100 μL / well (1 × 10⁶) into a 96-well cell culture plate. 5 Cells were seeded one cell per well and incubated at 37°C for 1 hour. The culture medium was then carefully removed and the plates were washed four times with PBS (plate washer procedure). Subsequently, CCK-8 reagent was prepared at a ratio of 10 μL per 100 μL of culture medium, thoroughly mixed, and then added to each 96-well cell culture plate at 100 μL / well. The plates were incubated at 37°C and 5% CO2 for 1 hour in a cell culture incubator. After incubation, the absorbance of the 96-well cell culture plate at 450 nm was measured using a microplate reader, and the results were recorded. The experimental results are shown in Figure 7, and it was confirmed that the Y109 molecule, which was a six-times repeat of the candidate fragment, had stronger adhesion-promoting activity towards NIH / 3T3 cells compared to the 019 fragment.

[0181] 3. Concentration-dependent activity of Y109 molecule in promoting NIH / 3T3 cell adhesion. Y109 molecules and controls (commercially available natural bovine collagen and commercially available recombinant humanized collagen) were diluted to 200 μg / mL with PBS to prepare a total of 255 μL of solution. After thorough mixing, 85 μL was added to 170 μL of PBS, and this process was repeated seven times to obtain a total of eight different concentrations. After dilution, 50 μL of each diluted solution was added to each 96-well cell culture plate to create three pores. The plates used were untreated on the culture surface. After incubation at 4°C overnight, the cell culture plates were washed once with PBS. Next, a 1% BSA solution was prepared and 100 μL was added to each well using a multichannel pipette, and incubated at room temperature for 1 hour. After removing the BSA solution, each well was washed once with 200 μL of PBS to remove the PBS. The cell density of NIH / 3T3 cells is 1 × 10⁻¹⁶ 6 Prepare the solution to cells / mL and use a multichannel pipette to transfer 100 μL / well (1 × 10⁶) into a 96-well cell culture plate. 5 Cells were seeded one cell per well and incubated at 37°C for 1 hour. After carefully removing the culture medium, the plates were washed four times with PBS (plate washer procedure). Subsequently, CCK-8 reagent was prepared at a ratio of 10 μL per 100 μL of culture medium, mixed, and then added to each 96-well cell culture plate at 100 μL / well. The plates were incubated at 37°C and 5% CO2 for 1 hour in a cell culture incubator. After incubation, the absorbance at 450 nm was measured using a microplate reader, and the results were recorded. The experimental results are shown in Figure 8, and it was confirmed that the NIH / 3T3 cell adhesion promoting activity by the Y109 molecule was enhanced with increasing concentration within a certain range.

[0182] In this specification, terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” or “several examples” mean that the specific features, structures, materials, or properties described in relation to the embodiment or example are included in at least one embodiment or example of the present invention. The suggestive use of these terms in this specification does not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or properties described may be appropriately combined and used in any one or more embodiments or examples, provided they do not conflict with each other.

[0183] Although embodiments of the present invention have been shown and described above, these embodiments are merely illustrative and do not limit the present invention. Those skilled in the art can modify, alter, substitute, and transform the above embodiments within the scope of the present invention.

Claims

1. A recombinant collagen monomer comprising at least one collagen-active fragment, The collagen-active fragment is characterized by comprising at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites, and is a recombinant collagen monomer.

2. The recombinant collagen monomer according to claim 1, characterized in that the collagen-active fragment is derived from the amino acids at positions 168 to 1196 of collagen.

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

4. The collagen-activating fragment is collagen Peptides from amino acid position 1089 to amino acid position 1172, and / or Peptides from amino acid position 234 to amino acid position 296, and / or Peptides from amino acid position 273 to amino acid position 311, and / or Peptides from amino acid position 288 to amino acid position 452, and / or Peptides from amino acid position 300 to amino acid position 485, and / or Peptides from amino acid position 444 to amino acid position 503, and / or Peptides from amino acid position 483 to amino acid position 512, and / or Peptides from amino acid position 501 to amino acid position 599, and / or Peptides from amino acid position 507 to amino acid position 686, and / or Peptides from amino acid position 591 to amino acid position 731, and / or Peptides from amino acid position 678 to amino acid position 806, and / or Peptides from amino acid position 726 to amino acid position 830, and / or Peptides from amino acid position 795 to amino acid position 833, and / or Peptides from amino acid position 798 to amino acid position 839, and / or Peptides from amino acid position 852 to amino acid position 866, and / or Peptides from amino acid position 828 to amino acid position 932, and / or Peptides from amino acid position 861 to amino acid position 995, and / or Peptides from amino acid position 927 to amino acid position 1094, and / or Peptides from amino acid position 987 to amino acid position 1103 The recombinant collagen monomer according to claim 1, characterized in that it is the same as described above.

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. Contains 2 to 12 collagen-active fragments, The collagen-active fragments may be the same or different. The recombinant collagen monomer according to claim 1, characterized in that the 2 to 12 collagen-active fragments are directly or indirectly bound to each other.

7. The recombinant collagen monomer according to claim 6, characterized by containing six collagen-active fragments.

8. Furthermore, it includes a stabilizing sequence, the stabilizing sequence being bound to the C-terminus of at least one collagen active fragment, The recombinant collagen monomer according to claim 1, characterized in that the amino acid sequence of the stabilizing sequence is sequence number 6.

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

25.

10. Recombinant collagen characterized by containing three recombinant collagen monomers according to any one of claims 1 to 9.

11. The recombinant collagen has a triple helix structure, The recombinant collagen monomers are of the same or different origins. The recombinant collagen according to claim 10, characterized in that the recombinant collagen monomers are bonded to each other by hydrogen bonds or electrostatic interactions.

12. A nucleic acid molecule characterized by encoding a recombinant collagen monomer according to any one of claims 1 to 9 or a recombinant collagen according to any one of claims 10 to 11.

13. A vector characterized by comprising the nucleic acid molecule described in claim 12.

14. Recombinant cells comprising the nucleic acid molecule described in claim 12 or the vector described in claim 13, or expressing the recombinant collagen monomer described in any one of claims 1 to 9 or the recombinant collagen described in any one of claims 10 to 11, The recombinant cells are eukaryotic or prokaryotic cells. The eukaryotic cells mentioned above are Pichia yeast, budding yeast, animal cells, or plant cells. Recombinant cells characterized in that the prokaryotic cells are Escherichia coli, Bacillus subtilis, or Bacillus licheniformis.

15. A method for producing recombinant collagen, A method for producing recombinant collagen, characterized by culturing the recombinant cells described in claim 14 under conditions suitable for protein expression.

16. The use of reagents in the manufacture of pharmaceuticals, The use is characterized in that the drug is used to improve collagen activity or to promote collagen expression or secretion, the reagent is used to activate collagen active fragments, and the collagen active fragments include at least three GER sites and / or GEK sites and / or RGD sites and / or GEN sites.

17. The use of reagents in the manufacture of pharmaceuticals, The use is characterized in that the drug is used to suppress collagen activity or to suppress collagen expression or secretion, the reagent is used to suppress collagen active fragments, and the collagen active fragments include at least three 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 is derived from the amino acids at positions 168 to 1196 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 collagen-activating fragment is collagen Peptides from amino acid position 1089 to amino acid position 1172, and / or Peptides from amino acid position 234 to amino acid position 296, and / or Peptides from amino acid position 273 to amino acid position 311, and / or Peptides from amino acid position 288 to amino acid position 452, and / or Peptides from amino acid position 300 to amino acid position 485, and / or Peptides from amino acid position 444 to amino acid position 503, and / or Peptides from amino acid position 483 to amino acid position 512, and / or Peptides from amino acid position 501 to amino acid position 599, and / or Peptides from amino acid position 507 to amino acid position 686, and / or Peptides from amino acid position 591 to amino acid position 731, and / or Peptides from amino acid position 678 to amino acid position 806, and / or Peptides from amino acid position 726 to amino acid position 830, and / or Peptides from amino acid position 795 to amino acid position 833, and / or Peptides from amino acid position 798 to amino acid position 839, and / or Peptides from amino acid position 852 to amino acid position 866, and / or Peptides from amino acid position 828 to amino acid position 932, and / or Peptides from amino acid position 861 to amino acid position 995, and / or Peptides from amino acid position 927 to amino acid position 1094, and / or Peptides from amino acid position 987 to amino acid position 1103 The use according to claim 16 or 17, characterized in that it is the same as the use according to claim 16 or 17.

21. Use of recombinant collagen monomers according to any one of claims 1 to 9 or recombinant collagen according to claim 10 or 11 in the manufacture of agents for cosmetic purposes, wound repair, or joint lubrication.

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

23. Use of a recombinant collagen monomer according to any one of claims 1 to 9 or recombinant collagen according to claim 10 or 11 for cosmetic purposes, wound repair, or joint lubrication.

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