Polypeptide, recombinant X-type humanized collagen and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a polypeptide, a recombinant X-type humanized collagen and application of the recombinant X-type humanized collagen. The invention provides a polypeptide which can effectively promote cell adhesion and improve cell proliferation activity. Specifically, the amino acid sequence of the polypeptide comprises n repetitive units; the amino acid sequence of the repetitive unit comprises any one of the following items (a)-(c): (a) a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; (b) a sequence obtained by adding, substituting and / or deleting one or more amino acid residues in the sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 and retaining a biological activity function; and (c) a sequence which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence as shown in SEQ ID NO.1 and retains a biological activity function. The invention provides a novel method for cartilage repair.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a polypeptide, recombinant humanized type X collagen and their applications, particularly to the polypeptide, recombinant humanized type X collagen and their application in cartilage repair. Background Technology

[0002] Collagen is the most abundant protein in the human body, accounting for 25%-35% of the total protein. It is widely distributed in connective tissue and has at least 28 subtypes, each distributed in different tissues.

[0003] Type X collagen is a short-chain, non-fibrous collagen. Its molecular structure consists of a homotrimer composed of three identical α1(X) chains. The triple helix domain is 132 nm long, only half the length of type II collagen, but it retains a larger non-collagenous globular domain at the C-terminus. (Human) COL10A1 The gene is located on the long arm of chromosome 6 (6q21-q22.3). Its coding region is highly compact and mainly distributed in two exons: one 169 bp exon encodes the signal peptide and the N-terminal non-collagenous domain, and the other approximately 2940 bp exon encodes the remaining N-terminal domain, the complete triple helix domain (463 amino acids), the C-terminal domain (161 amino acids), and the 3' untranslated region.

[0004] X-type collagen expression exhibits strict spatiotemporal specificity, being produced solely by mast chondrocytes in ossified regions of cartilage (such as the hypertrophic zone of the growth plate). Immunolocalization studies show that it presents a narrow banded distribution in the growth plates of human fetal long bones and ribs, closely overlapping with the calcification front. Of particular note is that X-type collagen synthesis precedes mineral deposition, suggesting its potential involvement in initiating the mineralization process. Specifically, in fetal cartilage, the immunoreactivity of X-type collagen precedes the calcium signal detected by Alizarin Red S, providing direct evidence for its pioneering role in the mineralization sequence.

[0005] The unique properties of type X collagen offer numerous possibilities for its applications in the medical field. In bone diseases, its specific expression pattern in the growth plate can serve as a molecular marker for endochondral ossification. In the intervertebral discs of osteoarthritis patients, changes in type X collagen expression may serve as a biomarker for disease progression or repair. Based on its gene regulatory mechanism, COL10A1 Cis-regulatory elements of genes can serve as intervention targets. In the field of regenerative medicine, the extraction and purification technology of X-type collagen has laid the foundation for the development of cartilage repair materials. Its controllable degradation characteristics (such as being reduced to 53 kDa by pepsin treatment) can be used to design biomaterials with specific release properties.

[0006] Therefore, it is necessary to explore effective ways to supplement or fill X-type collagen exogenously. Current technologies include methods for preparing X-type collagen peptides using animal-derived biomaterials (such as eggshell membrane extracts). However, because these are not human-derived, they may raise immunogenicity concerns when applied to bone tissue, thus limiting their use. Therefore, developing a non-immunogenic X-type collagen product suitable for large-scale production and with practical application potential has become an urgent research direction in this field. Summary of the Invention

[0007] One of the objectives of this invention is to provide a recombinant X-type humanized collagen that is highly safe and can effectively promote cell proliferation and adhesion.

[0008] This invention provides a polypeptide whose amino acid sequence comprises n repeating units, where n is an integer greater than or equal to 1. When n is an integer greater than or equal to 2, the repeating units are directly linked or linked by linking peptides (with one or more amino acid residues between each repeating unit); wherein the amino acid sequence of the repeating unit comprises any one of the following (a)-(c): (a) A sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; (b) A sequence in which one or more amino acid residues are added, substituted, and / or deleted, as shown in SEQ ID NO.1 or SEQ ID NO.2, while retaining biological activity; (c) A sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO.1 and retaining biological activity.

[0009] In the polypeptide of the present invention, the amino acid sequence of the repeating unit comprises a sequence in which 12-66 amino acid residues are added, substituted, and / or deleted (in some embodiments, the number of amino acid residues is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66) and the biological activity is retained. Preferably, the amino acid sequence of the biologically active functional sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence shown in SEQ ID NO. 16.

[0010] In the polypeptide of the present invention, the amino acid sequence of the repeating unit is extended by 1 to 54 integer amino acids at the N-terminus and / or C-terminus based on SEQ ID NO.16; preferably, it is extended by 1, 12, 48 or 54 amino acids.

[0011] In the polypeptide of the present invention, the amino acid sequence of the repeating unit is selected from any one of the following groups: (1) A sequence as shown in SEQ ID NO.3 that is extended by 54 amino acids at the N-terminus and 12 amino acids at the C-terminus based on SEQ ID NO.16, or a sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with it and retains biological activity. (2) A sequence as shown in SEQ ID NO.4 that extends 48 amino acids at the N-terminus and 12 amino acids at the C-terminus based on SEQ ID NO.16, or a sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with it and retains biological activity. (3) A sequence that extends 12 amino acids from the C-terminus of SEQ ID NO.16 as shown in SEQ ID NO.5, or a sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity and retains biological activity.

[0012] In the polypeptide of the present invention, n is an integer between 1 and 20 (in some embodiments, the number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), preferably an integer between 4 and 12; more preferably 4 or 12; In the polypeptide of the present invention, preferably, when n≥2, the repeating units are directly connected.

[0013] Preferably, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO. 6-10.

[0014] This invention utilizes synthetic biology and structural biology techniques to develop recombinant humanized type X collagen capable of forming a triple helix structure and performing the functions of human collagen. The aforementioned polypeptides can serve as monomers for recombinant humanized type X collagen. These polypeptides not only form recombinant humanized type X collagen with a triple helix structure, but the resulting recombinant humanized type X collagen also exhibits excellent performance in cell adhesion and cell proliferation promotion.

[0015] In some embodiments of the present invention, the substitution is a conserved amino acid substitution.

[0016] Conservative amino acid substitution refers to the replacement of one or more amino acids with amino acids that have similar or related properties.

[0017] In some embodiments of the present invention, the polypeptide is derived from human.

[0018] In some embodiments of the present invention, the polypeptide may form a triple helix structure.

[0019] In some embodiments of the present invention, the polypeptide has a flexible triple helix structure.

[0020] In some embodiments of the present invention, the polypeptide exists in the form of a trimer.

[0021] In some embodiments of the present invention, the triple helix structure of the polypeptide has one or more of the following activities or effects: cell adhesion activity, cell proliferation promotion activity.

[0022] The present invention also provides a fusion protein comprising the above-mentioned polypeptide; preferably, the polypeptide is obtained by fusing the polypeptide with other functional polypeptides.

[0023] Other functional peptides include, but are not limited to, other collagen peptides, preferably derived from truncated sequences of human collagen.

[0024] The present invention also provides recombinant humanized type X collagen, wherein the recombinant humanized type X collagen comprises the above-mentioned polypeptide; Preferably, the recombinant X-type humanized collagen has cell adhesion and / or cell proliferation-promoting activities. More preferably, it has biological activity against chondrocytes; Preferably, the recombinant X-type humanized collagen has a trimer and / or triple helix structure.

[0025] The aforementioned polypeptides can form trimers, which in turn form recombinant X-type humanized collagen with a triple helix structure.

[0026] In some embodiments, when the repeating unit of the polypeptide is mutated, the resulting polypeptide or recombinant humanized X-type collagen retains comparable or superior cell adhesion or cell proliferation-promoting activity compared to the polypeptide or recombinant humanized X-type collagen obtained from the unmutated repeating unit.

[0027] In some embodiments, when there are spacer sequences between the repeating units of the polypeptide, the obtained polypeptide or recombinant humanized X-type collagen has comparable or superior cell adhesion or cell proliferation-promoting activity compared to the polypeptide or recombinant humanized X-type collagen obtained by directly linking the repeating units.

[0028] In some embodiments, the recombinant X-type humanized collagen of the present invention is synthetic or recombinantly expressed.

[0029] The present invention also provides a polynucleotide encoding the above-mentioned polypeptide or fusion protein or recombinant humanized X-type collagen; Preferably, the polynucleotide comprises the nucleotide sequence shown in any one of SEQ ID NO. 11-15 or a degenerate sequence thereof.

[0030] The degenerate sequence refers to a nucleotide sequence obtained based on the degeneracy of the codon that is different from the nucleotide sequence shown in SEQ ID NO.11-15, but can still encode the aforementioned polypeptide, fusion protein, or recombinant humanized X-type collagen.

[0031] In some embodiments, the polynucleotide is codon-optimized for the host cell in which it is expressed.

[0032] In some implementations, the polynucleotide encoding recombinant humanized type X collagen can be operatively linked to expression control elements, such as promoters, terminators, and / or enhancers, to form a recombinant nucleic acid molecule or an expression cassette.

[0033] The polynucleotide of the present invention further comprises a sequence and / or a leader sequence encoding a purification tag to facilitate the purification or secretion of the polypeptide; preferably, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

[0034] The leader sequence includes, but is not limited to, sequences such as signal peptides.

[0035] The present invention also provides a biological material comprising the above-mentioned polynucleotides; the biological material is an expression cassette, a vector, or a host cell; The expression cassette may be a recombinant DNA molecule obtained by operably linking a control element upstream or downstream of the polynucleotide.

[0036] The vector includes plasmid vectors, viral vectors, transposons, artificial chromosomes, etc. The vector can be an expression vector or an integration vector. Preferably, the vector is an expression vector; the vector preferably further includes a control element operatively linked to the polynucleotide; the control element is preferably a promoter, terminator, and / or enhancer. Preferably, the host cell is a bacterium, fungus, or animal cell; wherein the bacteria preferably include Escherichia coli; the fungus preferably includes yeast, more preferably Saccharomyces cerevisiae or Pichia pastoris.

[0037] The present invention also provides a method for preparing the above-mentioned polypeptide or fusion protein or recombinant humanized X-type collagen, the method comprising: culturing host cells containing the above-mentioned polynucleotides, harvesting host cells and / or culture medium containing the polypeptide or the fusion protein or the recombinant humanized X-type collagen, and separating (purifying) the polypeptide or the fusion protein or the recombinant humanized X-type collagen therefrom.

[0038] In some embodiments of the present invention, the host cell is *Escherichia coli*. The host cell containing the polynucleotide is *E. coli* containing an expression vector carrying the polynucleotide. The expression vector may be a pET-28a series vector.

[0039] The above method may further include an enzyme cleavage tag step after isolating the polypeptide, the fusion protein, or the recombinant humanized X-type collagen.

[0040] As a specific embodiment, the method for constructing recombinant X-type humanized collagen of the present invention includes functional region screening and construction of production strains, large-scale bio-fermentation, protein induction expression, purification, and optional enzymatic digestion steps.

[0041] In some embodiments, the functional region screening and production strain construction steps include: (1) large-scale functional region screening to obtain the target gene functional region; (2) inserting the obtained target gene functional region into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid; (3) transforming the recombinant expression plasmid into Escherichia coli competent cells BL21(DE3) and screening to obtain positive Escherichia coli genetically engineered bacteria.

[0042] In some embodiments, the large-scale bio-fermentation step includes: inoculating the selected positive Escherichia coli genetically engineered bacteria into a shake flask containing a culture medium containing antibiotics, and culturing it in a constant temperature shaker at 220 rpm and 37°C.

[0043] In some embodiments, the protein induction expression steps include: (1) cooling the cultured shake flask to 16-30℃; (2) adding IPTG stock solution to induce expression; (3) placing the induced bacterial culture into a centrifuge bottle, centrifuging at 6000 rpm and 4℃ for 12 min, and then collecting the bacterial cells.

[0044] In some embodiments, the purification and optional enzymatic digestion steps of recombinant humanized type X collagen include: (1) crude purification of recombinant humanized type X collagen on a Ni affinity chromatography column; (2) enzymatic digestion with collagen tool enzymes in a certain proportion; and (3) purification of recombinant humanized type X collagen on an ion exchange column.

[0045] The present invention also provides compositions comprising one or more selected from the above-described polypeptides, fusion proteins, recombinant humanized X-type collagen, polynucleotides, and biomaterials.

[0046] The composition of the present invention is selected from biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone or cartilage regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, drugs or food additives; Preferably, the composition is a topical composition, an injectable composition, or an oral composition; Preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge, or fiber.

[0047] The polypeptides, fusion proteins, or recombinant humanized X-type collagen of the present invention can be prepared into compositions or kits.

[0048] In some embodiments, the composition or kit may be a composition or kit for tissue filling and / or volume enhancement and / or repair.

[0049] In some embodiments, the composition or kit may also contain auxiliary substances.

[0050] In some embodiments, the compositions of the present invention may be tissue repair agents / tissue fillers / tissue volume enhancers (the tissues include, but are not limited to, muscle tissue, tendons and ligaments, cartilage / bone tissue, etc.), comprising the aforementioned polypeptides or fusion proteins or recombinant type X humanized collagen. The compositions of the present invention may be injectable / implantable in the human body and do not elicit an immune response in the human body.

[0051] This invention also provides the application of the above-mentioned polypeptides, fusion proteins, recombinant X-type humanized collagen, polynucleotides, biomaterials or compositions in the preparation of bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, muscle or bone or cartilage or ligament regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, drugs or food additives.

[0052] The present invention also provides the use of the above-mentioned polypeptides, fusion proteins, recombinant X-type humanized collagen, polynucleotides, biomaterials or compositions in the preparation of products for any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement; preferably, tissue repair is cartilage repair.

[0053] The tissue repair includes, but is not limited to, skin tissue repair, wound repair, connective tissue (such as tendons or ligaments) repair, bone (hard bone) or cartilage repair, etc.; and the tissue filling / volume enhancement includes, but is not limited to, tissues from various parts of the human body.

[0054] The present invention also provides the application of the above-mentioned polypeptides, fusion proteins or recombinant X-type humanized collagen in any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement, and cosmetic surgery; preferably, the application is for non-disease diagnosis or treatment purposes.

[0055] Preferably, the tissue repair, tissue filling or volume enhancement, or cosmetic surgery is performed by injecting the polypeptide, the fusion protein, or the recombinant humanized X-type collagen.

[0056] The present invention also provides the use of the above-mentioned polypeptides, fusion proteins, recombinant humanized type X collagen, polynucleotides, biomaterials or compositions in the preparation of medicaments for the prevention and / or treatment of diseases or conditions related to type X collagen deficiency or for the supplementation of type X collagen.

[0057] The present invention also provides a method for promoting cell adhesion and / or proliferation and / or improving the extracellular matrix, the method comprising the step of contacting the above-mentioned polypeptide, fusion protein, recombinant humanized X-type collagen or composition with cells; the cells are preferably animal cells; more preferably mammalian cells; further preferably human cells; preferably, the method is for non-disease diagnosis or treatment purposes; Preferably, the promotion of cell adhesion and / or proliferation and / or improvement of the extracellular matrix occurs in vivo / in vitro.

[0058] The present invention provides a method for performing cosmetic surgery, tissue filling or augmentation, tissue repair, and ophthalmic treatment on subjects in need, the method comprising: administering the polypeptide, the fusion protein, or the recombinant humanized X-type collagen to the subject.

[0059] Preferably, the administration is oral or injectable.

[0060] Preferably, the subject is a human being.

[0061] The beneficial effects of this invention are at least as follows: This invention successfully expresses and prepares recombinant X-type humanized collagen, which possesses a triple helix structure and excellent biological activity. It can promote cell proliferation and cell adhesion, and can be used for medical or non-medical purposes, as well as for filling, volume enhancement, or repair in various human tissues and organs. Furthermore, the recombinant protein of this invention is derived from the human body and has 100% identical amino acid sequences to those of natural human collagen. When applied to the human body, it will not cause immune rejection or allergic reactions. It can also be produced on a large scale and efficiently, facilitating industrial preparation and practical application. Attached Figure Description

[0062] Figure 1 This is an electrophoresis image of rhX-1.

[0063] Figure 2 This is an electrophoresis image of rhX-2.

[0064] Figure 3 This is an electrophoresis image of rhX-3.

[0065] Figure 4 This is an electrophoresis image of rhX-4.

[0066] Figure 5 This is an electrophoresis image of rhX-5.

[0067] Figure 6 The results are for the circular dichroism UV scan of rhX-1.

[0068] Figure 7 The results are for the circular dichroism UV scan of rhX-5.

[0069] Figure 8 The peptide coverage results are for rhX-1, rhX-2, and rhX-3.

[0070] Figure 9 The results show the peptide coverage of rhX-4.

[0071] Figure 10 The results show the peptide coverage of rhX-5.

[0072] Figure 11The results show the cell adhesion activity of recombinant humanized X-type collagen.

[0073] Figure 12 Results of chondrocyte adhesion experiments using recombinant humanized X-type collagen.

[0074] Figure 13 The results of cell proliferation and toxicity testing for recombinant X-type humanized collagen.

[0075] Figure 14 The three-dimensional structure prediction result is for SEQ ID NO.1.

[0076] Figure 15 The three-dimensional structure prediction result is for SEQ ID NO.5.

[0077] In each diagram (if any), This means P < 0.01. This means P < 0.001. This means P < 0.0001. Detailed Implementation

[0078] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0079] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0080] Example 1: Construction and expression of recombinant X-type humanized collagen Step 1: Construction of recombinant X-type humanized collagen (1) Large-scale functional region screening of natural human type X collagen (UniProtKB / Swiss-Prot: Q03692.2) yielded the following different protein functional regions: GTRGPIGPPGIPGFPGSKGDPGSP (SEQ ID NO.1); GNMGPQGPKGIPGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLP GP KGDPGVGGPP (SEQ ID NO.2); Further investigation into variant sequences of the above sequence was conducted. Based on the sequence of SEQ ID NO.2, the sequence was extended backward or truncated to different lengths, retaining the "GPKGDPGVGGPP" (SEQ ID NO.16) sequence. Further screening yielded the following protein functional regions: GSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLP GPKGDPGVGGPP GLPGPVGPAGAK (SEQ ID NO.3); GPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLP GPKGDPGVGGPP GLPGPVGPAGAK (SEQ ID NO.4); GPKGDPGVGGPP GLPGPVGPAGAK (SEQ ID NO. 5).

[0081] To ensure the purification and stability of recombinant humanized X-type collagen, the amino acid fragments in the above repeating regions were optimized through multiple repetitions and direct ligation, resulting in recombinant collagen rhX-1, rhX-2, rhX-3, rhX-4, and rhX-5 (abbreviated as X-1, X-2, X-3, X-4, and X-5), with corresponding amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0082] The amino acid sequence of recombinant collagen rhX-1 (12 repeats of SEQ ID NO.1): GTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSPGTRGPIGPPGIPGFPGSKGDPGSP(SEQ ID NO.6)。

[0083] Amino acid sequence of recombinant collagen rhX-2 (4 repeats of SEQ ID NO.2): GNMGPQGPKGIPGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGNMGPQGPKGIPGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGNMGPQGPKGIPGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGNMGPQGPKGIPGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPP(SEQ ID NO.7)。

[0084] Amino acid sequence of recombinant collagen rhX-3 (4 repeats of SEQ ID NO.3): GSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAKGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAKGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAKGSHGLPGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAK (SEQ ID NO.8).

[0085] Amino acid sequence of recombinant collagen rhX-4 (4 repeats of SEQ ID NO.4): GPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAKGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAKGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAKGPKGETGPAGPAGYPGAKGERGSPGSDGKPGYPGKPGLDGPKGNPGLPGPKGDPGVGGPPGLPGPVGPAGAK (SEQ IDNO.9).

[0086] Amino acid sequence of recombinant collagen rhX-5 (12 repeats of SEQ ID NO.5): GPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGP KGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAKGPKGDPGVGGPPGLPGPVGPAGAK (SEQ IDNO.10).

[0087] Codon optimization was performed based on the E. coli expression system, and the corresponding nucleotide sequences encoding recombinant collagen rhX-1~rhX-5 are shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively.

[0088] Nucleic acid sequence of recombinant collagen rhX-1: GGAACAAGGGGGCCCATCGGTCCACCGGGTATCCCGGGCTTTCCGGGTTCCAAGGGTGATCCGGGAAGCCCAGGCACCCGTGGCCCGATCGGCCCACCGGGTATTCCGGGTTTTCCGGGTAGCAAAGGTGACCCTGGCTCGCCGGGGACCCGTGGTCCGATTGGTCCGCCTGGCATCCCGGGTTTCCCGGGCTCGAAAGGTGATCCGGGTTCTCCGGGTACTCGCGGTCCCATCGGCCCTCCGGGCATTCCGGGATTCCCGGGCAGCAAAGGTGACCCAGGTAGCCCGGGCACCCGTGGTCCGATTGGCCCACCGGGCATTCCGGGCTTCCCGGGCAGCAAAGGCGATCCGGGTTCTCCGGGCACCCGTGGTCCTATTGGTCCACCGGGCATCCCGGGCTTTCCGGGTTCCAAGGGCGACCCTGGTTCACCGGGCACCCGTGGCCCTATCGGTCCGCCGGGCATCCCGGGCTTCCCGGGTAGCAAGGGCGACCCGGGTTCGCCGGGCACCCGTGGTCCGATCGGCCCACCGGGTATCCCGGGATTCCCGGGTAGCAAAGGCGATCCGGGCTCTCCGGGAACCCGCGGCCCGATCGGTCCTCCGGGCATCCCGGGCTTCCCGGGTTCCAAGGGTGACCCGGGCTCCCCGGGTACACGCGGTCCGATCGGCCCACCGGGTATTCCGGGTTTCCCGGGCTCCAAGGGCGACCCGGGTAGCCCGGGTACGCGTGGTCCGATTGGTCCGCCAGGCATTCCGGGTTTTCCGGGTAGCAAGGGTGATCCGGGTAGCCCGGGTACGAGAGGTCCGATTGGCCCGCCGGGTATCCCGGGCTTTCCGGGTAGCAAAGGCGATCCGGGTAGCCCG (SEQ ID NO.11).

[0089] Nucleic acid sequence of recombinant collagen rhX-2: GGAAACATGGGGCCGCAGGGTCCGAAAGGTATCCCGGGAAGCCACGGCCTGCCGGGCCCGAAGGGCGAGACGGGTCCTGCGGGTCCGGCGGGTTACCCGGGCGCAAAAGGCGAGCGCGGTAGCCCGGGTAGCGATGGCAAACCGGGCTACCCGGGCAAACCGGGCCTGGATGGTCCGAAGGGTAATCCGGGTTTGCCTGGCCCGAAGGGTGATCCGGGTGTTGGTGGTCCACCGGGGAACATGGGCCCGCAGGGTCCGAAGGGGATTCCGGGCAGCCACGGCCTTCCGGGCCCGAAGGGCGAAACCGGTCCGGCGGGTCCGGCTGGTTATCCGGGTGCGAAAGGCGAGCGTGGTTCTCCGGGTTCTGATGGCAAGCCGGGTTATCCGGGTAAACCGGGACTGGATGGTCCAAAAGGCAACCCGGGTTTGCCGGGTCCGAAAGGTGACCCGGGCGTGGGCGGTCCGCCTGGCAACATGGGTCCGCAAGGTCCGAAGGGTATTCCGGGTAGCCATGGTCTGCCGGGACCGAAGGGCGAAACCGGTCCAGCTGGCCCAGCCGGTTACCCGGGTGCGAAAGGCGAGCGTGGTTCCCCGGGCAGCGACGGCAAACCGGGTTACCCGGGTAAACCGGGCCTGGACGGTCCCAAGGGCAATCCGGGCCTGCCGGGCCCCAAGGGCGACCCGGGTGTTGGTGGTCCGCCTGGCAATATGGGTCCCCAAGGTCCGAAGGGCATCCCGGGCTCCCATGGTTTACCGGGCCCGAAGGGCGAAACCGGTCCAGCGGGTCCGGCAGGCTACCCGGGCGCCAAAGGCGAACGTGGTTCGCCGGGCTCCGACGGCAAACCGGGCTATCCGGGCAAGCCGGGTTTGGACGGCCCAAAAGGCAACCCGGGTCTGCCTGGCCCAAAGGGTGATCCGGGTGTGGGTGGCCCGCCG (SEQ ID NO.12).

[0090] Nucleic acid sequence of recombinant collagen rhX-3: GGATCACACGGGCTTCCGGGACCGAAGGGTGAGACGGGTCCGGCAGGTCCGGCGGGTTACCCGGGTGCGAAAGGTGAACGTGGTAGCCCGGGTTCCGATGGTAAACCGGGTTATCCGGGCAAGCCGGGTTTGGACGGTCCGAAAGGAAACCCGGGCTTGCCGGGTCCGAAAGGTGATCCGGGCGTGGGCGGTCCACCGGGTCTGCCGGGACCAGTCGGACCGGCGGGTGCCAAAGGTAGCCACGGTCTGCCGGGTCCAAAGGGTGAAACCGGTCCGGCGGGTCCGGCAGGCTATCCGGGTGCGAAAGGCGAGCGTGGTTCGCCGGGCTCCGACGGCAAACCGGGCTACCCGGGCAAACCGGGCCTGGACGGCCCCAAGGGCAACCCGGGCCTGCCGGGTCCGAAGGGCGACCCTGGCGTTGGTGGTCCGCCTGGGCTGCCGGGCCCAGTTGGCCCGGCTGGCGCAAAAGGTAGCCACGGCTTGCCGGGCCCCAAGGGCGAAACCGGTCCGGCGGGCCCTGCGGGTTACCCGGGTGCGAAAGGTGAGCGCGGTTCTCCGGGTAGCGATGGCAAGCCGGGTTATCCGGGGAAGCCGGGCCTGGACGGTCCGAAAGGCAATCCGGGATTACCTGGACCGAAGGGCGATCCGGGGGTGGGTGGCCCACCGGGCCTGCCGGGTCCCGTGGGTCCAGCGGGTGCGAAAGGTAGCCATGGTCTCCCGGGCCCGAAGGGTGAAACCGGTCCAGCCGGCCCGGCTGGTTACCCGGGTGCAAAAGGCGAGCGTGGTAGCCCTGGTTCTGACGGTAAACCGGGTTACCCGGGCAAGCCGGGCCTGGATGGCCCTAAGGGCAACCCGGGTCTGCCGGGCCCGAAGGGCGATCCGGGCGTTGGTGGTCCACCCGGGTTGCCGGGTCCGGTGGGCCCAGCTGGCGCCAAG(SEQ ID NO.13)。

[0091] Nucleic acid sequence of recombinant collagen rhX-4: GGGCCCAAAGGAGAGACGGGTCCGGCAGGCCCGGCTGGCTATCCGGGCGCAAAAGGTGAGCGCGGTAGCCCGGGCTCCGATGGTAAGCCGGGATACCCGGGTAAACCGGGCTTGGACGGCCCGAAGGGCAACCCTGGTCTGCCGGGTCCGAAGGGCGATCCGGGTGTTGGTGGCCCTCCGGGCCTGCCGGGTCCGGTAGGCCCGGCAGGCGCGAAAGGTCCGAAAGGCGAAACCGGTCCGGCTGGTCCAGCCGGTTACCCGGGTGCAAAGGGCGAGCGTGGTAGCCCGGGCTCCGACGGTAAGCCTGGTTACCCGGGCAAACCGGGGCTGGATGGTCCGAAGGGTAACCCGGGCCTTCCGGGTCCGAAAGGTGATCCGGGCGTGGGCGGTCCACCGGGCCTGCCGGGCCCGGTGGGCCCAGCCGGTGCCAAGGGTCCGAAGGGTGAAACCGGTCCGGCGGGTCCGGCGGGTTATCCGGGCGCGAAAGGCGAGCGTGGTAGCCCGGGTAGCGACGGCAAGCCGGGCTACCCGGGTAAACCGGGCCTGGATGGTCCGAAAGGCAATCCGGGCCTGCCTGGTCCGAAAGGCGACCCGGGTGTTGGCGGTCCGCCTGGCCTCCCGGGTCCAGTGGGTCCGGCTGGCGCGAAGGGCCCGAAGGGCGAAACCGGTCCAGCGGGTCCGGCGGGTTACCCGGGTGCTAAAGGTGAACGTGGTTCTCCGGGTTCGGATGGCAAACCGGGTTATCCGGGCAAACCGGGTTTGGACGGCCCAAAGGGAAACCCGGGCTTGCCGGGTCCCAAGGGCGACCCGGGCGTCGGTGGTCCACCGGGTCTGCCAGGCCCGGTTGGTCCGGCGGGTGCGAAA (SEQ ID NO.14).

[0092] Nucleic acid sequence of recombinant collagen rhX-5: GGGCCCAAAGGAGACCCGGGTGTTGGCGGTCCGCCTGGCCTTCCGGGCCCGGTTGGTCCGGCTGGTGCAAAAGGTCCGAAGGGCGACCCGGGCGTGGGCGGTCCGCCGGGTCTGCCGGGTCCGGTTGGTCCGGCGGGTGCCAAAGGCCCCAAGGGCGACCCGGGTGTGGGCGGTCCACCGGGCTTGCCGGGCCCAGTGGGTCCGGCGGGTGCAAAAGGTCCGAAGGGCGACCCTGGCGTCGGTGGTCCGCCGGGCCTGCCGGGTCCGGTTGGCCCGGCAGGCGCGAAGGGTCCGAAAGGCGATCCGGGTGTCGGTGGCCCACCGGGTCTGCCGGGTCCGGTCGGTCCGGCGGGTGCGAAAGGCCCGAAAGGTGATCCGGGTGTAGGCGGTCCGCCTGGTTTACCGGGCCCGGTGGGTCCGGCAGGCGCGAAAGGCCCGAAGGGTGATCCGGGTGTTGGCGGCCCGCCTGGTCTGCCGGGTCCGGTTGGTCCGGCCGGTGCCAAGGGTCCGAAAGGTGATCCGGGCGTGGGCGGACCGCCAGGGCTGCCGGGTCCGGTTGGTCCGAAAGGCGACCCGGGCGTTGGTGGCCCACCGGGTCTGCCGGGTCCGGTGGGCCCGGCTGGCGCGAAAGGACCGAAAGGTGACCCTGGTGTTGGCGGTCCACCGGGCTTGCCGGGTCCGGTGGGCCCAGCGGGTGCGAAGGGTCCCAAGGGCGATCCGGGCGTGGGCGGTCCGCCGGGCTTGCCGGGCCCGGTGGGCCCTGCGGGTGCGAAAGGTCCGAAGGGCGATCCGGGTGTTGGCGGTCCACCGGGCCTGCCGGGCCCGGTGGGTCCGGCTGGTGCCAAG (SEQ ID NO.15).

[0093] Step 2 Expression of Recombinant Humanized Collagen Type X The gene functional regions (SEQ ID NO.11-15) synthesized in step 1 above were inserted into the pET-28a-Trx-His (GenScript Biotechnology Co., Ltd.) expression vector via KpnI and XhoI restriction sites to obtain the corresponding recombinant expression plasmids.

[0094] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process was as follows: (1) Take out the E. coli competent cells BL21(DE3) from the ultra-low temperature freezer and place them on ice. When they are half-thawed, take 2 μL of the plasmid to be transformed and add it to the E. coli competent cells BL21(DE3), and mix slightly 2-3 times. (2) Place the mixture on ice for 30 min, then heat shock it in a water bath at 42℃ for 45-90 s, and then place it on ice for 2 min. (3) Transfer it to a biosafety cabinet and add 700 μL of liquid LB medium, and then incubate it at 37℃ and 220 rpm for 60 min. (4) Take 200 μL of bacterial solution and spread it evenly on an LB plate containing kanamycin sulfate. (5) Incubate the plate in a 37℃ incubator for 15-17 h until uniformly sized colonies grow.

[0095] Pick 5-6 single colonies from the transformed LB agar plates and place them in a shake flask containing an antibiotic stock solution (100 mg / L ampicillin). Incubate the flasks at 220 rpm and 37°C in a constant temperature shaker for a certain period of time until they become misty. Then, cool the shake flasks to 16-30°C, add IPTG (0.5 mM) to induce expression for a period of time, aliquot the bacterial culture into centrifuge bottles, centrifuge at 6000 rpm and 4°C for 12 min, collect the bacterial cells, and record the cell weight.

[0096] The collected bacterial cells were resuspended in a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). The bacterial suspension was cooled to ≤15°C and homogenized twice or sonicated to disrupt the cells. After this process, the bacterial suspension was collected. The disrupted bacterial suspension was aliquoted into centrifuge bottles and centrifuged at 17,000 rpm at 4°C for 30 min. The supernatant was collected for subsequent purification.

[0097] Purification methods include: 1. Crude purification of the target protein (based on affinity chromatography principle) 1.1 Column Balancing Operation An affinity chromatography column (Ni-NTA column) was used, and the column material was equilibrated using an equilibration buffer. The equilibration buffer consisted of 200 mM sodium chloride, 25 mM Tris (tris(hydroxymethyl)aminomethane), and 20 mM imidazole. During equilibration, the flow rate was controlled at 10 mL / min, and equilibration was continued until the pH of the column eluent matched that of the equilibration buffer, ensuring that the column material was in a suitable state for binding to the target protein.

[0098] 1.2 Sample loading procedure The supernatant containing the target protein is slowly added to the chromatography column after equilibration in step 1.1. The loading flow rate is controlled at 5 mL / min. After the supernatant has completely flowed through the chromatography column, the specific binding of the target protein to the column material is completed.

[0099] 1.3 Cleaning procedure for contaminating proteins Add 100 mL of washing buffer to the chromatography column. The washing buffer has the same composition as the equilibration buffer in step 1.1 (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). Control the washing flow rate to 10 mL / min. After the washing buffer has completely flowed through the chromatography column, remove non-specifically bound proteins from the column material.

[0100] 1.4 Elution and Detection Procedures for Target Protein Add 20 mL of elution buffer to the chromatography column, which consists of 200 mM sodium chloride, 25 mM Tris, and 250 mM imidazole. Control the elution flow rate at 10 mL / min and collect the flow-through liquid (i.e., the solution containing the target protein with the tag sequence) during the elution process simultaneously.

[0101] 1.5 Column Recycling Operation After the target protein has been eluted, add 1M imidazole working solution to the chromatography column and control the washing flow rate to 10mL / min to remove the residual protein components on the column and complete the column regeneration process for subsequent reuse.

[0102] 2. Enzymatic digestion of the target protein 2.1 Collagen tool enzyme enzymatic digestion reaction Take the flow-through solution containing the target protein collected in step 1.4, and add TEV enzyme to the flow-through solution at a mass ratio of 20:1 between the total amount of the target protein and the total amount of collagen tool (TEV enzyme, tobacco etch virus protease); place the mixed solution in a constant temperature environment of 16℃ for enzyme digestion reaction, and the enzyme digestion reaction lasts for 2 hours to achieve specific cleavage of the target protein tag sequence.

[0103] 2.2 Dialysis desalination treatment Transfer the protein mixture after enzyme digestion in step 2.1 to a dialysis bag. Place the dialysis bag in dialysis solution (20mM sodium chloride, 20mM Tris) at 4°C for initial dialysis for 2 hours. Then replace the dialysis solution with fresh solution and place the dialysis bag in an environment of 4°C for overnight dialysis (dialysis time not less than 12 hours) to remove imidazole and some small molecule impurities from the mixture. At the same time, adjust the buffer environment of the protein solution to prepare for subsequent purification.

[0104] 3. Purification of the target protein (based on the principle of ion exchange chromatography) 3.1 Column Balancing Operation An anion exchange chromatography column (Capto Q, Cytiva) was used to equilibrate the column material with solution A, which consisted of 20 mM Tris and 20 mM sodium chloride. The equilibration flow rate was controlled at 10 mL / min, and equilibration was continued until the conductivity of the column eluent was consistent with that of solution A, ensuring that the column material was in a low-salt environment suitable for the binding of impurities and proteins.

[0105] 3.2 Sample loading and flow-through collection Add the dialyzed protein solution from step 2.2 to the ion exchange chromatography column equilibrated in step 3.1, and control the loading flow rate at 5 mL / min. During sample loading, simultaneously collect the flow-through liquid (the target protein does not bind to the column material and flows out with the flow-through liquid, while other proteins specifically bind to the column material) and collect it as QFL (Quaternary Fractionation Liquid). Perform electrophoresis detection on the collected QFL (e.g., SDS-PAGE electrophoresis). Store the qualified flow-through liquid containing the target protein at 4°C to avoid protein denaturation.

[0106] 3.3 Elution of Impurities After the sample loading in step 3.2 is completed, solution B is added to the ion exchange chromatography column. Solution B consists of 1M sodium chloride and 20mM Tris. The elution flow rate is controlled at 10mL / min, and the column is continuously washed for 5 column volumes (CV) to completely elute the impurities bound to the column.

[0107] 3.4 Column Cleaning Operation After the impurities have been eluted, the chromatography column is cleaned with a suitable cleaning solution (such as an aqueous solution containing 0.5M sodium hydroxide or a special regeneration solution for the corresponding ion exchange column) to remove residual impurities on the column material, thus completing the cleaning and regeneration of the column material for reuse in subsequent experiments.

[0108] The purified recombinant humanized type X collagen (QFL in step 3.2) was detected by SDS-PAGE. The electrophoresis results are shown in the figure. Figures 1 to 5 This shows that recombinant collagen rhX-1 to rhX-5 (X-1 to X-5) were successfully expressed and prepared.

[0109] The theoretical molecular weights of the purified recombinant collagen proteins X-1, X-2, X-3, X-4, and X-5 were 26.2 KD, 28.5 KD, 27.9 KD, 25.8 KD, and 24.2 KD, respectively. The apparent molecular weights after purification were largely consistent with the theoretical molecular weights, with slight deviations in the bands due to the positive and negative charges inherent in the proteins. During expression, expression was initiated using the inducing agent IPTG. The host RNA polymerase bound the promoter, transcribing the target gene into mRNA. Ribosomes then bound the mRNA, translating it into a polypeptide chain. After enzymatic digestion, the tagged fusion protein was cleaved, and subsequent purification yielded the target protein. The target protein bands appeared in positions close to the theoretical molecular weights. In the QFL (quantitative fibroblast flow lysate), X-1, X-2, X-3, X-4, and X-5 proteins with good purity were observed. These processes ultimately yielded the target protein with high purity.

[0110] Example 2: Circular dichroism UV-Vis analysis of recombinant X-type humanized collagen 1.1 Experimental Methods (1) Instrument parameter settings Bandwidth: 1.0nm; Step: 1.0nm; Measurement range: 190-260nm (far-UV region scanning) / 250-340nm (near-UV region scanning); Time per point: 0.5s; Repeats: 3 times; Cell Length: 10mm | 0.5mm; Temperature: Room temperature.

[0111] (2) Near and far ultraviolet scanning of standard samples The scanning wavelength was set to 180-340 nm for background testing and blank buffer testing. Then, the circular dichroism near and far ultraviolet absorption of 1 mg / mL camphorsulfonic acid (CSA) standard solution was collected in the wavelength range of 180-340 nm.

[0112] (3) Sample processing Take protein samples and concentrate them to a protein concentration of 1 mg / mL using a 10 kDa ultrafiltration concentrator (Millibo).

[0113] (4) Far-ultraviolet scanning of samples Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a far-ultraviolet scan at 190-260 nm according to the above parameters and collect the data.

[0114] (5) Near-ultraviolet scanning of samples Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a near-ultraviolet scan at 250-340 nm according to the above parameters and collect the data.

[0115] (6) Scanning image processing All scanned spectra were processed using the software Pro-Data Viewer to perform baseline subtraction and smoothing.

[0116] (7) Experimental results and analysis The results are shown below. Figure 6 and Figure 7 Among the five recombinant X-type humanized collagens mentioned above, rhX-1 and rhX-5 both showed positive peaks at 220-230 nm, indicating that they all possess a triple helix structure.

[0117] Example 3: Peptide coverage of recombinant type X humanized collagen Detecting the coverage of recombinant X-type humanized collagen peptides can verify the consistency between the product and the target sequence, confirm whether it is the target protein, assess its structural integrity, and determine whether it has been degraded or has lost a sequence. It ensures the stability of protein function and is a key indicator for ensuring the correctness and quality of the product in research and development and quality control. High coverage (such as 94%~100%) reflects the reliability of quality.

[0118] (1) Protein sample preparation: Take the purified humanized collagen sample, add an appropriate amount of denaturation buffer (containing urea or guanidine hydrochloride), and denature it at high temperature (such as 95℃) to fully unfold the protein; then add dithiothreitol (DTT) to reduce the disulfide bond, and then use iodoacetamide (IAA) for alkylation treatment to block free thiol groups and prevent protein refolding.

[0119] (2) Enzymatic hydrolysis: Dilute the treated protein solution with ammonium bicarbonate buffer to a denaturant concentration of less than 1M, add sequencing grade trypsin (or other specific proteases, such as Lys-C), mix at an enzyme:protein ratio of 1:50 to 1:100, and incubate at 37°C for 12 to 16 hours to allow the protein to be enzymatically hydrolyzed into small peptides.

[0120] (3) Peptide extraction and desalting: After the enzymatic hydrolysis reaction is completed, formic acid is added to terminate the reaction (final concentration of about 0.1%), and the supernatant is collected by centrifugation; the peptides are desalted using a C18 solid phase extraction column to remove buffer salts and impurities, and then vacuum dried and reconstituted with 0.1% formic acid aqueous solution.

[0121] (4) LC-MS / MS analysis: The reconstituted peptide sample was injected into a high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The peptides were separated by a reversed-phase column (such as a C18 column), and the primary and secondary mass spectrometry data of each peptide were obtained by mass spectrometry.

[0122] (5) Data analysis and coverage calculation: The mass spectrometry data is matched with the theoretical amino acid sequence database of the target collagen (using software such as MaxQuant and Proteome Discoverer), and the proportion of the detected peptide sequence in the full-length sequence of the target protein is calculated, i.e. peptide coverage (calculation formula: coverage = number of detected amino acid residues / total number of amino acid residues in the protein × 100%).

[0123] (6) Experimental results and analysis: The peptide coverage results of rhX-1, rhX-2, and rhX-3 are as follows: Figure 8 As shown in the figure. The peptide coverage results for rhX-4 and rhX-5 are as follows. Figure 9 and Figure 10 As shown.

[0124] Example 4: Detection of cell adhesion activity of recombinant X-type humanized collagen A collagen adhesion layer was prepared in a 96-well plate, and then HFB cells (human fibroblast cells) were seeded into the 96-well plates. In this experimental system, the negative control was Dulbecco's Phosphate-Buffered Saline (D-PBS), and the positive control was the international standard bovine collagen type I (BCL I). The correlation between negative and positive values ​​was analyzed to determine whether the assay system was functioning correctly. The D-PBS mainly consisted of added calcium ions (Ca). 2+ ) and magnesium ions (Mg 2+The PBS buffer used in this study chelates non-specific adhesion molecules. Collagen provides a high-quality extracellular matrix environment for cells, helping them adhere to cell culture plates. The higher the protein's adhesiveness, the faster it helps cells adhere, resulting in a greater number of cells remaining in the wells after washing. The number of cells remaining in the wells can be quantitatively analyzed using the CCK8 (Cell Counting Kit-8) kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), allowing for the calculation of the collagen-induced cell adhesion ratio. This value further reflects collagen's cell adhesion-promoting effect.

[0125] The specific steps are as follows: (1) Sample preparation: Bovine type I collagen was diluted to 1 mg / mL using D-PBS buffer and set as a positive control (PC); the negative control (NC) was D-PBS buffer; the collagen experimental group was also set to 1 mg / mL; and cell-free complete culture medium was set as the zeroing well.

[0126] (2) Coating: Add collagen experimental samples, positive control and negative control to the microplate, 100 μL per well, 5 replicates per group, and incubate overnight at 4 ℃.

[0127] (3) Blocking: Discard the supernatant, add 100 μL of 1% BSA (Bovine Serum Albumin; heat-inactivated at 56℃ for 30 min), and incubate at 37℃ for 60 min. Discard the supernatant and wash 3 times with D-PBS solution.

[0128] (4) Cell seeding: Add 10 to each well 5 Cells in good culture condition resuspended in D-PBS were incubated at 37 °C for 120 min. Each well was washed three times with D-PBS solution.

[0129] (5) Detection: Add 100 μL of serum-free medium containing 5 μL of CCK8 reaction solution to each well and incubate at 37 ℃ for 120 min. Detect using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. Simultaneously, prepare a blank control well containing 5 μL of CCK8 reaction solution but without cells.

[0130] (6) Calculate the cell adhesion degree according to the following formula. The cell adhesion rate reflects the cell adhesion force of collagen. The higher the cell adhesion force, the better the external environment can be provided to the cells in a short time, which helps the cells adhere.

[0131] ; In the formula: P: Relative cell adhesion ratio; OD1: The average UV absorbance of each pore of the test sample at 450 nm; OD2: The average UV absorbance of each replicate well of the negative control sample at 450 nm; OD0: The average UV absorbance of each replicate well in the blank control group at 450 nm.

[0132] (7) Statistical analysis: The statistical difference between the target recombinant humanized collagen and the negative control was analyzed using a two-tailed t-test.

[0133] See results Figure 11 As shown, compared with the D-PBS group (NC), bovine type I collagen (PC) and recombinant humanized collagen rhX-1, rhX-2, rhX-3, rhX-4, and rhX-5 also significantly promoted cell adhesion.

[0134] Example 5: Chondrocyte adhesion experiment of recombinant X-type humanized collagen The cell adhesion activity of recombinant type X humanized collagen was detected using human chondrocyte C28 / I2 cells. The specific experimental method was as described in Example 4. The negative control was Dulbecco's phosphate-buffered saline (D-PBS); the positive control was bovine type I collagen. Both the positive control group and the collagen experimental group were prepared at a concentration of 1 mg / mL.

[0135] The results are as follows Figure 12 As shown, rhX-1, rhX-2, rhX-3, rhX-4, and rhX-5 (X-1~X-5) exhibited a promoting effect on the adhesion of human chondrocyte C28 / I2. Compared with the NC group (100%), the relative cell adhesion activity of the PC group was 135%, indicating that the positive control had an adhesion effect, verifying the effectiveness of this experiment. The relative cell adhesion activities of the rhX-2 group (158%) and the rhX-4 group (168%) were higher than those of the PC group, while the relative cell adhesion activities of the rhX-1, rhX-3, and rhX-5 groups were as high as 205%, 185%, and 202%, respectively, which were significantly higher than those of the PC group, and had a significant effect on promoting the adhesion of human chondrocyte C28 / I2.

[0136] Example 6: Cell proliferation and toxicity assays of recombinant type X humanized collagen CCK8 reagent contains WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt), which is reduced by cellular dehydrogenases to a highly water-soluble yellow formazan dye by the electron carrier 1-methoxy-5-methylphenazineonium sulfate (1-Methoxy PMS). The amount of formazan dye produced is directly proportional to the number of viable cells. Therefore, this property can be used to directly analyze the proliferation and toxicity of human chondrocyte C28 / I2.

[0137] The specific steps are as follows: (1) Experimental grouping: The culture medium containing 0.4% serum was the negative control (NC), 8% NaCl was the positive control (PC), the recombinant X-type humanized collagen with a concentration of 1 mg / mL was the experimental group, and the complete culture medium without cells was the zeroing well.

[0138] (2) Sample preparation: Dilute the sample to the working concentration with 0.4% serum culture medium, filter it through a 0.22 µm microporous membrane for sterilization, and use it directly as a sample. Dissolve NaCl in 0.4% serum culture medium to a final concentration of 8%, and filter it for sterilization.

[0139] (3) Cell seeding: When the cell confluence reaches approximately 90%, digest and count the cells. Digest and count the cells at a rate of (5-10) × 10⁶ cells per well. 3 Cells were seeded in 96-well plates, and the edge wells were blocked with 100 µL of PBS. Cells were cultured for 24 h to allow adherence.

[0140] (4) Starvation treatment: After the cells adhered, the supernatant was aspirated, and 100 µL of culture medium containing 0.4% serum was added to each well and cultured for 24 h.

[0141] (5) Cell drug administration: Discard the supernatant and replace the corresponding group solutions in sequence, with 4 replicates per group, and culture for 24 h.

[0142] (6) Experimental detection: Discard the culture medium, add 100 µL (including 5 µL CCK8 solution) of basal culture medium, incubate in an incubator for 3 h, and detect using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The cell viability calculation formula is as follows.

[0143] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%; In the formula: As: Absorbance of experimental wells (including cells, culture medium, CCK8 solution and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK8 solution, but excluding drugs); Ab: Absorbance of the blank group (including culture medium and CCK8 solution, but excluding cells and drugs).

[0144] (7) Statistical analysis: The statistical differences between the samples and the negative control were analyzed using a two-tailed t-test.

[0145] See results Figure 13 The results showed that, compared with the negative control group, the cell survival rate of the positive control group was significantly reduced, and the difference was statistically significant.

[0146] Experiments have shown that the five recombinant humanized collagen proteins X-1, X-2, X-3, X-4 and X-5 (rhX-1~rhX-5) in the experimental group not only have no cytotoxicity, but also have significant cell proliferation-promoting activity.

[0147] Example 7: Prediction of the three-dimensional structure of recombinant X-type humanized collagen The protein structure prediction software AlphaFold2 was used to predict the complex structure of the three strands of the above sequences SEQ ID NO.1 and SEQ ID NO.5. The results are shown below. Figure 14 and Figure 15 This shows that the above sequences can form stable triple helix structures, which is consistent with the excellent biological activity verified above.

[0148] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide contains n repeating units, where n is an integer greater than or equal to 1. When n is an integer greater than or equal to 2, the repeating units are directly linked or linked by linking peptides; wherein the amino acid sequence of the repeating unit contains any one of the following (a)-(c): (a) A sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; (b) A sequence in which one or more amino acid residues are added, substituted, and / or deleted, as shown in SEQ ID NO.1 or SEQ ID NO.2, while retaining biological activity; (c) A sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO.1 and retaining biological activity.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the repeating unit comprises a sequence in which 12-66 amino acid residues are added, substituted and / or deleted in the sequence shown in SEQ ID NO.2 while retaining biological activity. Preferably, the amino acid sequence of the biologically active functional sequence has at least 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence shown in SEQ ID NO.

16.

3. The polypeptide according to claim 2, characterized in that, The amino acid sequence of the repeating unit is extended by an integer number of amino acids from the N-terminus and / or C-terminus of SEQ ID NO. 16; preferably, it is extended by 1, 12, 48 or 54 amino acids.

4. The polypeptide according to claim 3, characterized in that, The amino acid sequence of the repeating unit is selected from any one of the following groups: (1) A sequence as shown in SEQ ID NO.3 that is extended by 54 amino acids at the N-terminus and 12 amino acids at the C-terminus based on SEQ ID NO.16, or a sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with it and retains biological activity. (2) A sequence as shown in SEQ ID NO.4 that extends 48 amino acids at the N-terminus and 12 amino acids at the C-terminus based on SEQ ID NO.16, or a sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with it and retains biological activity. (3) A sequence that extends 12 amino acids from the C-terminus of SEQ ID NO.16 as shown in SEQ ID NO.5, or a sequence that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity and retains biological activity.

5. The polypeptide according to any one of claims 1 to 4, characterized in that, n is an integer between 1 and 20, preferably an integer between 4 and 12; more preferably 4 or 12. Preferably, when n≥2, the repeating units are directly connected.

6. The polypeptide according to any one of claims 1 to 5, characterized in that, The polypeptide has an amino acid sequence as shown in any one of SEQ ID NO. 6-10.

7. A fusion protein, characterized in that, The fusion protein comprises the polypeptide according to any one of claims 1 to 6; preferably, it is obtained by fusing the polypeptide with other functional polypeptides.

8. Recombinant X-type humanized collagen, characterized in that, The recombinant X-type humanized collagen comprises the polypeptide according to any one of claims 1 to 6; Preferably, the recombinant X-type humanized collagen has cell adhesion and / or cell proliferation promotion activities; more preferably, it has biological activity against chondrocytes. Preferably, the recombinant X-type humanized collagen has a trimer and / or triple helix structure.

9. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide according to any one of claims 1 to 6, the fusion protein according to claim 7, or the recombinant X-type humanized collagen according to claim 8; Preferably, the polynucleotide comprises the nucleotide sequence shown in any one of SEQ ID NO. 11-15 or a degenerate sequence thereof.

10. The polynucleotide according to claim 9, characterized in that, The polynucleotide further comprises a sequence encoding a purification tag and / or a leader sequence; preferably, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

11. A biomaterial, characterized in that, The biomaterial comprises the polynucleotide as described in claim 9 or 10; The biomaterial is an expression cassette, vector, or host cell; Preferably, the vector is an expression vector; the vector preferably further comprises a control element operatively linked to the polynucleotide; the control element is preferably a promoter, terminator, and / or enhancer. Preferably, the host cell is a bacterium, fungus, or animal cell; wherein the bacteria preferably include Escherichia coli; the fungus preferably includes yeast, more preferably Saccharomyces cerevisiae or Pichia pastoris.

12. A method for preparing the polypeptide according to any one of claims 1 to 6, the fusion protein according to claim 7, or the recombinant humanized X-type collagen according to claim 8, characterized in that, The preparation method includes: culturing host cells containing the polynucleotides of claim 9 or 10, harvesting host cells and / or culture medium containing the polypeptide or the fusion protein or the recombinant humanized X-type collagen, and isolating the polypeptide or the fusion protein or the recombinant humanized X-type collagen therefrom.

13. A composition, characterized in that, The composition comprises one or more selected from the polypeptides of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant X-type humanized collagen of claim 8, the polynucleotide of claim 9 or 10, and the biomaterial of claim 11.

14. The composition according to claim 13, characterized in that, The composition is selected from biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone or cartilage regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, drugs or food additives. Preferably, the composition is a topical composition, an injectable composition, or an oral composition; Preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge, or fiber.

15. The use of the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant X-type humanized collagen of claim 8, the polynucleotide of claim 9 or 10, the biomaterial of claim 11, or the composition of claim 13 or 14 in the preparation of bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, muscle or bone or cartilage or ligament regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, pharmaceutical or food additives.

16. The use of the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant X-type humanized collagen of claim 8, the polynucleotide of claim 9 or 10, the biomaterial of claim 11, or the composition of claim 13 or 14 in the preparation of products for any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement; preferably, tissue repair is cartilage repair.

17. The use of the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, or the recombinant X-type humanized collagen of claim 8 in any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement, and cosmetic surgery; the use is not for disease diagnosis or treatment purposes.

18. The use of the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant humanized X-type collagen of claim 8, the polynucleotide of claim 9 or 10, the biomaterial of claim 11, or the composition of claim 13 or 14 in the preparation of a medicament for the prevention and / or treatment of diseases or conditions related to X-type collagen deficiency or for the supplementation of X-type collagen.

19. A method for promoting cell adhesion and / or proliferation and / or improving the extracellular matrix, characterized in that, The method includes the step of contacting a cell with the polypeptide of any one of claims 1 to 6, the fusion protein of claim 7, the recombinant type X humanized collagen of claim 8, or the composition of claim 13 or 14; the cell is preferably an animal cell; more preferably a mammalian cell; and even more preferably a human cell; the method is not for disease diagnosis or treatment purposes. Preferably, the promotion of cell adhesion and / or proliferation and / or improvement of the extracellular matrix occurs in vivo / in vitro; the improvement of the extracellular matrix refers to promoting the formation of a network structure of the extracellular matrix.

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