A recombinant humanized type III collagen molecule and preparation method and application thereof

Abstract

The application discloses a kind of recombinant III type humanized collagen molecule and its preparation method and application, belong to genetic engineering technical field.The amino acid sequence of the recombinant III type humanized collagen is designed by the molecular parent nucleus serial combination of as shown in SEQ ID NO.1~9.This application's recombinant III type humanized collagen is compared with natural collagen, molecular weight is determined, can be expressed in preparation in eukaryotic cell such as yeast, has the biological activity of being superior to natural collagen and commercially available same type recombinant collagen, can effectively promote cell multiplication, adhesion, migration;It has wide application prospect in the fields such as drug, medical device, biomaterial, tissue engineering, cosmetics or health care product.This application's recombinant III type humanized collagen amino acid sequence is selected from natural collagen amino acid sequence, and production host cell does not contain endotoxin, has better security.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a recombinant type III humanized collagen molecule, its preparation method, and its application. Background Technology

[0002] Collagen is an important structural protein and a vital component of the human body. It accounts for 25%-30% of the total protein in the human body, with 90% found in the skin and bones. Collagen comprises approximately 95% of the dermal connective tissue, about 80% of bone, and a high 85% in tendons. An adult human body contains approximately 3 kilograms of collagen, widely distributed throughout various tissues, playing a crucial role in maintaining the shape, structure, and function of skin and organs. Based on tissue location, physiological function, and molecular structure, collagen is mainly classified into 28 types. Types I, II, and III account for about 90% of all collagen, and these three have been the most extensively studied and are now used in industrial applications. Type III is often referred to as "infant collagen" because it constitutes 80% of infant skin, gradually decreasing with age, with adult skin containing only about 20% type III collagen. Type III collagen can provide nutrients to cells, directly bind to angiocytes to promote the formation of new blood vessels, and has whitening, anti-wrinkle, repairing and soothing effects. Its value has been demonstrated in skin care products, medical aesthetics and serious medical fields.

[0003] Currently, commercially available collagen is primarily natural collagen, mostly produced using animal tissues such as cattle, pigs, and fish as raw materials, through acid, alkali, and enzymatic hydrolysis to obtain collagen derivatives. Collagen obtained in this way is mostly used in skincare and health supplements, but its immunogenicity, allergenicity, and risk of carrying viruses limit its application in the medical field. Although companies specializing in natural collagen extraction, such as Shuangmei and Chuang'er Biotechnology, possess mature animal collagen extraction technology and quality systems, applying animal-derived collagen to injectable aesthetic medicine, two major problems hinder the sustained large-scale production of animal collagen. First, to strictly control the risk of carrying viruses (mad cow disease, foot-and-mouth disease), the raw materials used in pharmaceuticals must be traceable, requiring complete feeding and medication records from animal birth to slaughter, resulting in a shortage of qualified animals. Second, the immunogenicity of animal-derived collagen is uncertain, affected by factors such as purity, structure, donor, extraction method, implantation site, and cross-linking method, leading to poor safety controllability, which is also a key focus of drug regulatory authorities. Based on the above objective reasons, collagen products that are homologous to human tissues, have low immunogenicity, no viral risk, diverse molecular functions, and are affordable are more in line with user value needs.

[0004] Recombinant collagen expression technology has opened up new possibilities for the industrial application of human collagen. By optimizing the sequence and synthesizing genes, the full-length or fragmented human collagen can be introduced into industrial protein expression systems, such as mammalian cells, insect cells, yeast, E. coli, and spores, allowing for large-scale production of recombinant collagen through scalable culture. Currently, humanized recombinant collagen produced from E. coli and yeast has been successfully applied in cosmetics and injectable aesthetic medicine, and its application continues to expand in serious medical and health product fields, showing promising prospects. Among the two mainstream microbial expression strains, E. coli intracellular expression requires cell lysis, has a high content of extracellular proteins leading to high production costs, and its cell wall components contain large amounts of endotoxins, peptidoglycans, and other pyrogens, posing safety risks. Therefore, Pichia pastoris is gradually becoming the mainstream production strain. Pichia pastoris is a GRAS (Generally Regard As Safe) strain, enabling extracellular secretion expression without endotoxin risks. It can be fermented at high density on a large scale, yields high amounts, and has a clear genetic background, making it an ideal host for collagen expression. However, Pichia pastoris also has two major limitations in protein expression. First, there are multiple proteases inside and outside the cell that degrade recombinant collagen peptides, making it impossible to obtain a single molecule. Second, most recombinant collagen molecules cannot be effectively expressed and secreted in Pichia pastoris, requiring sophisticated molecular design, secretion strategy design, and high-throughput screening methods.

[0005] Therefore, molecules with good activity, stability, and heterologous expression, along with efficient production strains, high-density fermentation, and product separation and purification processes, are key to achieving low-cost manufacturing of recombinant humanized collagen for large-scale production and application. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant type III humanized collagen molecule, its preparation method, and its application, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a recombinant type III humanized collagen, wherein the amino acid sequence of the recombinant type III humanized collagen is designed by tandem combination of molecular cores as shown in SEQ ID NO.1~9.

[0008] The second technical solution of the present invention is a nucleic acid molecule encoding the recombinant III humanized collagen, the nucleotide sequence of which is shown in SEQ ID NO.19~27.

[0009] The third technical solution of the present invention is a recombinant vector containing the nucleic acid molecule.

[0010] The fourth technical solution of the present invention is a host cell containing the recombinant vector.

[0011] The fifth technical solution of the present invention is a method for preparing the recombinant III humanized collagen, comprising the following steps: The recombinant III humanized collagen is expressed using the nucleic acid molecule, the recombinant vector, or the host cell, and then isolated and purified.

[0012] The sixth technical solution of the present invention is a composition comprising the recombinant III humanized collagen and excipients.

[0013] The seventh technical solution of the present invention is the application of the recombinant III humanized collagen or the composition in the preparation of drugs, medical devices, biomaterials, tissue engineering, cosmetics or health products.

[0014] The eighth technical solution of the present invention is a drug, medical device, biomaterial, tissue engineering, cosmetic or health product, comprising the recombinant III humanized collagen or the composition thereof.

[0015] Based on the above technical solution, the present invention has the following technical effects: The recombinant type III humanized collagen prepared by this invention has advantages over other fragments of human type III collagen as molecular core assembly molecules in terms of protein expression level, molecular stability, and cell activity. Since the molecular core is derived from the natural human type III collagen sequence, it will not produce an immune rejection reaction when applied to the human body. At the same time, the production method is simple and stable, and low-cost manufacturing of recombinant type III humanized collagen can be achieved. Attached Figure Description

[0016] Figure 1 Electrophoresis images of recombinant humanized type III collagen (SEQ ID NO. 10-18) induced by Pichia pastoris; where M is the marker, 1 is purified recombinant humanized type III collagen (SEQ ID NO. 10), 2 is purified recombinant humanized type III collagen (SEQ ID NO. 11), 3 is purified recombinant humanized type III collagen (SEQ ID NO. 12), 4 is purified recombinant humanized type III collagen (SEQ ID NO. 13), 5 is purified recombinant humanized type III collagen (SEQ ID NO. 14), 6 is purified recombinant humanized type III collagen (SEQ ID NO. 15), 7 is purified recombinant humanized type III collagen (SEQ ID NO. 16), 8 is purified recombinant humanized type III collagen (SEQ ID NO. 17), and 9 is purified recombinant humanized type III collagen (SEQ ID NO. 18). Detailed Implementation

[0017] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0018] This invention provides a recombinant type III humanized collagen, wherein the amino acid sequence of the recombinant type III humanized collagen is designed by tandem combination of molecular cores as shown in SEQ ID NO.1~9.

[0019] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 1.

[0020] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 2.

[0021] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 3.

[0022] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 4.

[0023] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 5.

[0024] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 6.

[0025] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 7.

[0026] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 8.

[0027] In some specific implementations, the core sequence of the recombinant type III humanized collagen molecule is shown in SEQ ID NO. 9.

[0028] In some specific implementations, the amino acid sequence of the recombinant III humanized collagen is shown in SEQ ID NO.10~18.

[0029] This invention also provides a homologous molecular core sequence assembly scheme to obtain recombinant type III humanized collagen molecules.

[0030] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from a homologous molecular nucleus sequence, as shown in SEQ ID NO. 10.

[0031] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from a homologous molecular nucleus sequence, as shown in SEQ ID NO. 11.

[0032] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from a homologous molecular nucleus sequence, as shown in SEQ ID NO. 12.

[0033] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from a homologous molecular nucleus sequence, as shown in SEQ ID NO. 13.

[0034] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from a homologous molecular nucleus sequence, as shown in SEQ ID NO. 14.

[0035] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from the same molecular nucleus sequence, as shown in SEQ ID NO. 15.

[0036] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from the same molecular nucleus sequence, as shown in SEQ ID NO. 16.

[0037] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from the same molecular nucleus sequence, as shown in SEQ ID NO. 17.

[0038] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from the same molecular nucleus sequence, as shown in SEQ ID NO. 18.

[0039] Embodiments of the present invention also provide a nucleic acid molecule encoding the recombinant III humanized collagen, the nucleotide sequence of which is shown in SEQ ID NO.19~27.

[0040] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 10. The nucleic acid molecule sequence is shown in SEQ ID NO. 19.

[0041] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 11. The nucleic acid molecule sequence is shown in SEQ ID NO. 20.

[0042] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 12. The nucleic acid molecule sequence is shown in SEQ ID NO. 21.

[0043] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 13. The nucleic acid molecule sequence is shown in SEQ ID NO. 22.

[0044] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 14. The nucleic acid molecule sequence is shown in SEQ ID NO. 23.

[0045] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 15. The nucleic acid molecule sequence is shown in SEQ ID NO. 24.

[0046] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 16. The nucleic acid molecule sequence is shown in SEQ ID NO. 25.

[0047] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 17. The nucleic acid molecule sequence is shown in SEQ ID NO. 26.

[0048] In some specific embodiments, the nucleic acid molecule encodes recombinant type III humanized collagen SEQ ID NO. 18. The nucleic acid molecule sequence is shown in SEQ ID NO. 27.

[0049] In some specific implementations, the recombinant type III humanized collagen encoded by the nucleic acid molecule has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO. 10, and retains the cell proliferation-promoting effect of SEQ ID NO. 10.

[0050] This invention also provides a recombinant vector containing the nucleic acid molecule.

[0051] In some specific implementations, the carrier is pPIC9K.

[0052] A vector is a nucleic acid fragment encoding a recombinant protein that can be transferred to a recipient cell, where it can be stably inherited and fulfill the functions of transcription, translation, and synthesis of the recombinant protein. Gene expression vectors typically contain an antibiotic resistance marker gene, an autonomously replicating sequence, and a recombinant protein expression cassette (promoter, multiple cloning site, terminator). Vectors are classified into free vectors and integrative vectors. Commonly used expression vectors for *E. coli* include the pET series. In addition, many artificially constructed expression vectors suitable for microorganisms, yeast, plants, and mammalian cells have been developed. Commonly used genome-integrating expression vectors for Pichia pastoris protein expression systems include pPIC9, pPIC9K, pPICZa, pGAPZa, the pPichiaPink series, and pAO815. In some scenarios, the CRISPR gene editing system can also be used to integrate the expression cassette of the target gene into the *Pichia pastoris* genome to achieve the expression of the target gene. In this embodiment, the carrier is pPIC9K-SEQ ID NO. 10, pPIC9K-SEQ ID NO. 11, pPIC9K-SEQ ID NO. 12, pPIC9K-SEQ ID NO. 13, pPIC9K-SEQ ID NO. 14, pPIC9K-SEQ ID NO. 15, pPIC9K-SEQ ID NO. 16, pPIC9K-SEQ ID NO. 17, or pPIC9K-SEQ ID NO. 18.

[0053] This invention also provides a host cell containing the recombinant vector.

[0054] In some specific implementations, the host cell is a eukaryotic cell or a prokaryotic cell.

[0055] In some specific implementation schemes, the eukaryotic cells are Penicillium, Aspergillus niger, Trichoderma, Aspergillus oryzae, Pichia pastoris, Kluyveromyces martensii, and Yersinia lipophila; the prokaryotic cells are Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens.

[0056] The term "host cell" refers to any cell type that can be adapted to the transformation, transfection, and transduction of the protein expression vector and gene editing vector described in this application. "Host cell" encompasses any offspring of the parent cell, which may differ from the parent cell due to mutations occurring during replication. The host cell can be any cell useful in the production of recombinant type III humanized collagen as described in this application. To produce recombinant collagen, the gene encoding recombinant type III humanized collagen can be synthesized into one or more vectors, and then the vector can be transformed into a host cell. The gene for recombinant type III humanized collagen can exist in the host cell cytoplasm as a free plasmid, or it can be integrated into the host cell genome using homologous or non-homologous recombination to obtain transformants and transformed cells that are not identical to the parent cell. Methods for introducing the vector into the host cell are well-known, such as electroporation, transfection, microinjection, gene gun technology, liposome-mediated transformation, etc.

[0057] In some specific implementations, the host cell is Pichia pastoris, Bacillus, Escherichia coli, or mammalian cells.

[0058] This invention also provides a method for preparing the recombinant III humanized collagen, comprising the following steps: The recombinant III humanized collagen is expressed using the nucleic acid molecule, the recombinant vector, or the host cell, and then isolated and purified.

[0059] In some specific implementations, hydrophobic interaction, cation-anion exchange chromatography, or mixed hydrophobic-cation chromatography methods are preferred for protein purification.

[0060] In some specific implementation schemes, the N-terminal redundant sequence of recombinant type III humanized collagen can be digested with enzymes, preferably Kex2 / Ste13 / TEV protease.

[0061] In some specific implementation schemes, the host cells are subjected to pure culture and fermentation. The culture medium and culture conditions are well known to those skilled in the art. The expression method can be induced expression, with inducing factors including temperature, pH, available carbon sources, and unavailable carbon sources; or it can be constitutive expression, which is accompanied by the growth and division process of the host cells. The expression elements used in constitutive expression are well known to those skilled in the art.

[0062] The embodiments of the present invention also provide the activity of the recombinant III humanized collagen in promoting cell proliferation, migration and adhesion, and its use in related products.

[0063] This invention also provides a composition comprising the recombinant III humanized collagen and excipients.

[0064] The present invention also provides the use of the recombinant III humanized collagen or the composition in the preparation of pharmaceuticals, medical devices, biomaterials, tissue engineering, cosmetics or health products.

[0065] This invention also provides a drug, medical device, biomaterial, tissue engineering, cosmetic or health product, including the recombinant III humanized collagen or the composition.

[0066] This invention provides a recombinant type III humanized collagen molecule nucleus SEQ ID NO.1, and based on this nucleus, adjusts the amino acid sequences at both ends to obtain eight molecular nucleus units SEQ ID NO.2-9. These nine nucleus units SEQ ID NO.1-9 are tandemly linked to form a multi-copy molecule, thus obtaining a series of recombinant humanized collagen molecules. The tandem linkage can be of the same molecular nucleus: such as (SEQ ID NO.1)n, where n is an integer greater than or equal to 1, and when n is greater than or equal to 2, the repeating sequences are directly connected; the tandem linkage can also be of different molecular nucleus: such as (SEQ ID NO.1 + SEQ ID NO.2 + …)n, where n is an integer greater than or equal to 1, and when n is greater than or equal to 2, the repeating sequences are directly connected; optionally, the N-terminus of the recombinant type III humanized collagen contains an amino acid sequence that can be cleaved by Kex2 / Ste13 / TEV proteases.

[0067] Further, in the above-mentioned recombinant type III humanized collagen molecule, the recombinant type III humanized collagen molecule comprises: a) the amino acid sequence shown in SEQ ID NO. 10 and its proline hydroxylation sequence; b) an amino acid sequence and its corresponding proline hydroxylation sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO. 10, and retaining the cell proliferation-promoting effect of SEQ ID NO. 10; c) one or more amino acids added, substituted, deleted or inserted in the amino acid sequence of SEQ ID NO. 10 and their corresponding proline hydroxylation sequences, and retaining the cell proliferation-promoting effect of SEQ ID NO. 10; or d) an amino acid sequence encoded by a nucleotide sequence, wherein the nucleotide sequence is the same as the amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO. 10, and the amino acid sequence retains the cell proliferation-promoting effect of SEQ ID NO. 10.

[0068] This invention also provides an assembly scheme of two or more parent nucleus sequences to obtain recombinant type III humanized collagen molecules.

[0069] In some specific implementations, the recombinant type III humanized collagen is a protein sequence assembled from ≥2 molecular core sequences, and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO.10, and retains the cell proliferation-promoting effect of SEQ ID NO.10.

[0070] Sequence identity refers to the "percentage of amino acid sequence identity" relative to a reference polypeptide sequence. This is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence, introducing vacancies where necessary to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways well known to those skilled in the art, such as BLAST, ALIGEN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0071] In the above text, addition refers to the addition of one or more amino acids to the C-terminus or N-terminus of the recombinant type III humanized collagen amino acid sequence. Insertion refers to the insertion of one or more amino acid residues at appropriate positions in the recombinant type III humanized collagen amino acid sequence; the inserted amino acid residues may be all or partly adjacent to each other or not adjacent to each other. Deletion refers to the deletion of one or more amino acids from the recombinant type III humanized collagen amino acid sequence. Substitution refers to the conservative substitution of one or more amino acids in the recombinant type III humanized collagen amino acid sequence. Additions, insertions, and deletions do not occur in the molecular core sequences SEQ ID NO. 1-9, and the number of additions can be 2-11.

[0072] The aforementioned variants of recombinant type III humanized collagen molecules must be modified while retaining the same / equivalent cell adhesion properties as the protein fragment SEQ ID NO. 10.

[0073] The variant of the above-mentioned recombinant type III humanized collagen molecule can be an amino acid sequence obtained by conservative substitution of amino acids in the sequence of SEQ ID NO. 10 according to Table 1.

[0074] Table 1

[0075] In the above text, all amino acids in the polypeptide sequence are L-type amino acids. One or more of these amino acids (e.g., 2-5) can be replaced with D-type amino acids, artificially modified amino acids, or rare amino acids found in nature to improve the bioavailability, stability, and / or antiviral activity of the polypeptide. D-type amino acids refer to amino acids corresponding to the common L-type amino acids that make up proteins; artificially modified amino acids refer to common L-type amino acids that make up proteins after modification such as methylation, phosphorylation, or amidation; rare amino acids found in nature include uncommon amino acids that make up proteins and amino acids that do not make up proteins, such as hydroxyproline, 5-hydroxylysine, methylhistidine, and homoserine.

[0076] In the specific implementation schemes described above, recombinant proteins refer to DNA fragments encoding specific protein sequences ligated to specific expression vectors using recombinant DNA technology. These expression vectors are then transformed into specific host cells to obtain recombinant strains, which can efficiently synthesize recombinant proteins under certain culture conditions. Protein expression systems include prokaryotic expression systems, such as *Escherichia coli*, *Bacillus*, and *Corynebacterium glutamicum*; eukaryotic expression systems, such as yeast, filamentous fungi, and mammalian cells (CHO, HEK293); and insect expression systems. Furthermore, protein expression systems also include cell-free expression systems. Cell-free expression systems represent a revolutionary breakthrough in in vitro protein synthesis technology, and their rapid, flexible, and controllable characteristics make them an important supplement to traditional cell expression systems. With decreasing costs, increased yields, and expanded functionality, they will play a more central role in biomedical research and development, synthetic biology, and in vitro diagnostics, especially in "emergency biomanufacturing" (such as responding to emerging infectious diseases) and "complex protein research," where they are expected to become one of the mainstream technologies. Recombinant collagen refers to collagen prepared through gene recombination technology. Artificially designed DNA sequences encoding collagen are introduced into host cells using methods known to those skilled in the art, allowing them to exist stably within the cells. This can occur as free plasmids or integrated into the genome. Large-scale production of recombinant collagen is achieved through amplified culture. Human collagen is classified into more than 28 types based on tissue location, physiological function, and molecular structure. Types I, II, and III collagen are the most studied, with Type I and Type III being the two most abundant collagen components in the skin. Type I collagen is in the form of bundles, supporting the skin structure and maintaining its resilience; while Type III collagen is more like a fine mesh, scattered around Type I, tightly binding dermal cells and water, determining the skin's elasticity and smoothness. Type I and Type III collagen constitute the extracellular matrix network, supporting organs, protecting the body, and are also involved in cell attachment and migration. The recombinant type III humanized collagen provided by this invention has a highly consistent amino acid sequence with human type III collagen, exhibits good cell proliferation, migration, and adhesion activity, and has good tissue compatibility.

[0077] Example 1 Recombinant Type III Humanized Collagen Sequence Design The maternal gene was designed to be UniProt ( https: / / www.uniprot.org / uniprotkb / The website obtained the α1 chain protein sequence of natural human type III collagen (reference sequence number P02461), and the mature peptide sequence is 1466 amino acids in length. Based on the fragment position, secondary structure type, charge, hydrophilicity / hydrophobicity, amino acid distribution, and other physicochemical properties, a recombinant type III humanized collagen molecular core SEQ ID NO. 1 was designed. Further adjustments to the C-terminal and N-terminal lengths yielded recombinant type III humanized collagen molecular cores SEQ ID NO. 2-9, which serve as assembly units for the recombinant type III humanized collagen molecule.

[0078] Table 2

[0079] Example 2 Gene synthesis of recombinant type III collagen The amino acid sequences of recombinant type III humanized collagen (SEQ ID NO. 10-18) were optimized according to the codon preference of Pichia pastoris, yielding the corresponding nucleotide coding sequences (SEQ ID NO. 19-27). The complete gene synthesis was performed by Qingke Biotechnology Co., Ltd., and the recombinant plasmid vectors were named pPIC9K-SEQ ID NO. 10, pPIC9K-SEQ ID NO. 11, pPIC9K-SEQ ID NO. 12, pPIC9K-SEQ ID NO. 13, pPIC9K-SEQ ID NO. 14, pPIC9K-SEQ ID NO. 15, pPIC9K-SEQ ID NO. 16, pPIC9K-SEQ ID NO. 17, and pPIC9K-SEQ ID NO. 18.

[0080] SEQ ID NO.10:。

[0081] SEQ ID NO.11:。

[0082] SEQ ID NO.12: ERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPG MRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRG。

[0083] SEQ ID NO.13: GAPPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAA GEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGAPGPAGPRGAAGEPGRDGVPGGPGMRGMP。

[0084] SEQ ID NO.14:。

[0085] SEQ ID NO.15: GERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGP GMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGERGAPGPAGPRGAAGEPGRDGVPGGPGMRMR.

[0086] SEQ ID NO.16: GERGAPGPAGPRGAAGEPGRDGVPGGP

[0087] SEQ ID NO.17: AGERGAPGPAGPRGAAGEPGRDGVPGGPGMAGERGAGP

[0088] SEQ ID NO.18: AGERGAPGPAGPRGAAGEPGRDGVPGGAGERGAPGPAGPRGAAGEPGRDGVPGGAGERGAPGPAGPRGAAGEPGRDGVPGGAGERGAPGPGPAGPRGAAGEPGRDGVPGGAGERGAPGPGPAGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPRGAAGEPGRDGVPGGAGERGAPGPGPGPRGAAGEPGRPG

[0089]

[0090]

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[0093]

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[0098] Example 3 Transformation of Pichia pastoris First, the recombinant plasmid was linearized using a SacI restriction endonuclease kit (ThermoFisher). The reaction system was performed according to the kit instructions. The digestion temperature was 37℃, and the reaction time was at least 2 hours. After the digestion reaction, the linearized plasmid was recovered using a DNA purification kit (Sigma), yielding the linear plasmid fragment for transformation.

[0099] Mix 3-5 μg of linearized pPIC9K-SEQ ID NO. 10, pPIC9K-SEQ ID NO. 11, pPIC9K-SEQ ID NO. 12, pPIC9K-SEQ ID NO. 13, pPIC9K-SEQ ID NO. 14, pPIC9K-SEQ ID NO. 15, pPIC9K-SEQ ID NO. 16, pPIC9K-SEQ ID NO. 17, and pPIC9K-SEQ ID NO. 18 plasmids with 80 μL of Pichia pastoris competent cells, transfer to electroporation cuvettes spaced 0.2 cm apart, electroporate once at 1.5 kV, 25 μF, 200 Ω, immediately resuspend the cells in 1M pre-cooled sorbitol solution, incubate at 30°C for 1-2 h, centrifuge to collect the cells, and plate on MD agar plates (1 L contains 13.4 g of Pichia pastoris plasmids). YNB (0.2g biotin, 20g glucose, 20g agar powder) is cultured at 30℃ for 2-3 days until transformants grow.

[0100] Example 4 Screening of high-yield recombinant type III humanized collagen strains The probability of Pichia pastoris producing ≥2 copies of transformants in a single transformation is about 1%. Therefore, to obtain high-copy transformants, colonies on MD plates were spotted onto YPD medium (1L containing 10 g yeast extract, 20 g tryptone, 20 g glucose, and 2 g agar powder) plates with G418 concentrations of 0.5 g / L, 1 g / L, and 2 g / L, respectively. If the transformant could grow on the high-concentration G418 YPD plate, it indicated that the transformant contained multiple copies of the gene, that is, multiple fragments were integrated into the Pichia pastoris genome. The high-copy transformants obtained after screening can be used as high-yield recombinant type III humanized collagen strains.

[0101] Example 5 Fermentation of recombinant type III humanized collagen The production strain was inoculated into 50 mL of YPD medium and cultured at 30°C with shaking at 220 rpm for 24 h. Then, it was transferred to 500 mL of YPD liquid medium and cultured at 30°C with shaking at 220 rpm for another 24 h as a secondary seed culture. A 10% (v / v) inoculum was transferred to a 10 L fermenter containing 5 L of sterilized BSM basal salt medium (1 L of which contains 40 g glycerol, 18.2 g potassium sulfate, 26.7 mL phosphate, 0.93 g calcium sulfate dihydrate, 14.9 g magnesium sulfate, and 4.13 g potassium hydroxide). Fermentation was initiated at a growth temperature of 25°C, pH 5.5, and dissolved oxygen maintained at approximately 20%. After a rapid increase in dissolved oxygen and depletion of low-sugar levels, glycerol feeding was initiated at a rate of 80 mL / h, maintaining dissolved oxygen at approximately 20% throughout the process. When the wet weight of the fermentation broth reached 140-180 g / L, feeding was stopped for 1 h to deplete the glycerol, and then methanol was added to induce fermentation. The methanol flow rate was initially increased to 10 mL / h during the induction phase, and then increased to 50 mL / h after 3 h. Fermentation was terminated after 90 h of induction.

[0102] Example 6 Purification of recombinant type III humanized collagen The fermentation broth was centrifuged at 4000 rpm to collect the supernatant, which was then filtered through a 0.45 μm plate membrane to further remove bacterial cells. The supernatant was then collected. Ultrafiltration (UF) was performed using a 10 kDa molecular weight cutoff device for desalting and decolorization. Ultrafiltration was stopped when the conductivity of the solution decreased to 100 μs / cm, and the final volume of the ultrafiltration solution was maintained at 1 / 3-1 / 5 of the initial volume. Purification was then performed using a cation exchange resin with a loading volume of 3 column volumes. Elution was performed using a solution of 50 mM sodium acetate, 1 M sodium chloride, and pH 4.5. The target protein was collected based on the UV detector signal. The electrophoresis results of the purified recombinant type III humanized collagen are shown below. Figure 1 As shown, M is the molecular weight marker, and 1 to 9 are the purified recombinant type III humanized collagen SEQ ID NO.10-19, respectively.

[0103] Example 7 Assay for cell proliferation / cytotoxicity of recombinant type III humanized collagen (MTT assay) Human skin fibroblasts in logarithmic growth phase (HSF) were seeded in 96-well plates (5 × 10⁻⁶ cells / well). 3(Each well) was incubated at 37℃ in 5% CO2 for 24 h. Fresh culture medium containing the corresponding concentration of the sample was added to the sample groups, while the normal group and blank control group were replaced with fresh culture medium and incubated at 37℃ in 5% CO2 for another 24 h. After incubation, MTT solution was added to each well, and incubation continued for 4 h. The culture medium was removed, DMSO solution was added, and the mixture was shaken to mix. The absorbance value (OD490) at 490 nm was measured.

[0104] .

[0105] Table 3. Recombinant type III humanized collagen promotes cell proliferation / cytotoxicity

[0106] According to the test results (Table 3), the cell viability of the recombinant type III humanized collagen SEQ ID NO. 10-19 sample group was not significantly different from that of the normal control group (100%) in human skin fibroblasts (HSF) within the concentration range of 3.12 mg / mL, revealing that the recombinant type III humanized collagen SEQ ID NO. 10-19 sample had no cytotoxicity within the concentration range of 3.12 mg / mL.

[0107] Example 7 Evaluation of the cell adhesion-promoting activity of recombinant type III humanized collagen The sample was prepared to the desired concentration and added at 500 μL / well to a 48-well cell culture plate, then dried at room temperature for 1 h. 500 μL of mouse embryonic fibroblasts (NIH / C3T3, cell density 6.2 × 10⁻⁶) were added to each well. 4 ( / well), 3 biological replicates, cultured at 37℃, 5% CO2 for 2 h. After gentle rinsing with D-Hanks solution, separation was performed using 200 μL of 0.5% Triton X-100 solution. The supernatant was collected, and lactate dehydrogenase activity was detected using a lactate dehydrogenase kit. Statistical results are expressed as mean ± SD and analyzed using SPSS software. Independent samples t-tests were used for statistical analysis between the two groups. The statistical results were defined as follows: α = 0.05, and p < 0.05 (compared to the normal control group) was considered statistically significant.

[0108] ; ; In the formula, A: OD440 absorbance; C standard: standard solution concentration 0.2 μmol / L; Cpr: sample protein concentration, gprot / mL (prot refers to protein); R: lactate dehydrogenase activity.

[0109] Table 4. Cell adhesion-promoting activity of recombinant type III humanized collagen

[0110] Cell adhesion refers to the contact and interaction between cells and adjacent cells through direct or indirect means. This interaction includes direct contact via cell surface and indirect contact via the extracellular matrix. Cell adhesion assays can be used to detect the effects of different treatments (growth factors, drugs, gene manipulation) on cell adhesion ability and are widely used to assess the adhesion of various cell types to the extracellular matrix. In this experiment, a sample concentration of 1.56 mg / mL was used. The results are shown in Table 4. The cell adhesion-promoting activity of recombinant type III humanized collagen (SEQ ID NO. 10-19) was significantly increased compared to the blank control group, with an increase of 16.7%-36.6%. Compared with the positive control human placental type III collagen and commercially available recombinant type III collagen, (SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 17, and SEQ ID NO. 18) showed significant advantages in promoting cell adhesion activity.

[0111] Example 9 Evaluation of the cell migration-promoting activity of recombinant type III humanized collagen Mouse 3T3 cells in the logarithmic growth phase were used as samples and seeded in 12-well plates (3 × 10⁻⁶ cells per well). 5 Cells (cells / well) were cultured at 37℃ in 5% CO2 for 24 h. Scratches were applied, followed by two gentle washes with PBS. Fresh culture medium was added to the normal control group, while fresh culture medium containing the corresponding concentration of the sample was added to the sample group. This was repeated three times biologically, and the cells were cultured at 37℃ in 5% CO2 for another 24 h. Images were taken at 0 h and 24 h after drug administration, and the scratch area was recorded. ImageJ software (https: / / imagej.nih.gov / ij) was used to process the cell migration images and analyze the healing rate of the scratched areas. Initial scratch area and cell-free blank area data were obtained, and the migration rate was calculated. Statistical results are expressed as mean ± SD and analyzed using SPSS software. An independent samples t-test was used for statistical analysis between the two groups. The statistical results were considered statistically significant with α = 0.05 as the test cutoff, and p < 0.05 (compared to the normal control group).

[0112] .

[0113] Table 5. Cell migration-promoting activity of recombinant type III humanized collagen

[0114] Cellular repair capacity is a crucial characteristic for cells to maintain tissue integrity and function. Under physiological conditions, cells can repair damaged tissues through various mechanisms such as proliferation and migration. Cell migration plays a key role in the healing process of damaged tissues. Microfilaments form elongated pseudopodia in front of the cell via actin and contract behind the cell via myosin-mediated migration, providing the driving force for cell migration. This experiment used mouse embryonic fibroblasts to establish a scratch assay model to evaluate whether the samples possess repair efficacy. At a sample concentration of 1.56 mg / mL, recombinant humanized type III collagen (SEQ ID NO. 10-19) exhibited significant cell migration-promoting activity, with an enhancement range of 19.4%-36.2%. Under the same conditions, the enhancement rates of the positive control human placental type III collagen and commercially available recombinant type III collagen were 21.4% and 17.7%, respectively, indicating that recombinant humanized type III collagen (SEQ ID NO. 10-19) has significant cell migration-promoting activity and revealing that this sample of recombinant humanized type III collagen (SEQ ID NO. 10-19) has repair efficacy.

[0115] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A recombinant III humanized collagen, characterized in that, The amino acid sequence of the recombinant type III humanized collagen is designed by tandem combination of molecular cores as shown in SEQ ID NO.1~9.

2. The recombinant III humanized collagen according to claim 1, characterized in that, The amino acid sequence of the recombinant III humanized collagen is shown in SEQ ID NO.10~18.

3. A nucleic acid molecule encoding the recombinant III humanized collagen as described in claim 1 or 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.19~27.

4. A recombinant vector comprising the nucleic acid molecule of claim 3.

5. A host cell, characterized in that, It includes the recombinant vector of claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is Pichia pastoris, Bacillus, Escherichia coli, or mammalian cell.

7. A method for preparing the recombinant III humanized collagen as described in claim 1 or 2, characterized in that, Includes the following steps: The recombinant III humanized collagen is expressed using the nucleic acid molecule of claim 3, the recombinant vector of claim 4, or the host cell of claim 5, and then isolated and purified.

8. A composition, characterized in that, Includes the recombinant III humanized collagen and excipients as described in claim 1 or 2.

9. The use of the recombinant III humanized collagen of claim 1 or 2 or the composition of claim 8 in the preparation of pharmaceuticals, medical devices, biomaterials, tissue engineering, cosmetics or health products.

10. A drug, medical device, biomaterial, tissue engineering, cosmetic, or health product, characterized in that, Includes the recombinant III humanized collagen of claim 1 or 2 or the composition of claim 8.

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