Recombinant humanized type ii collagen and uses for cartilage repair

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant humanized type II collagen and a use of cartilage repair. The recombinant humanized type II collagen has good chondrocyte adhesion activity, good biocompatibility and low immunogenicity; experiments show that the protein can effectively promote chondrocyte adhesion and migration, and inhibit the expression of inflammatory factors, and is suitable for cartilage repair and other tissue engineering applications.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the use of recombinant humanized type II collagen in cartilage repair. Background Technology

[0002] Cartilage, a crucial component of human joints, primarily fulfills the core functions of cushioning pressure, reducing friction during movement, and maintaining joint stability. Its unique avascular and nerve-free anatomical structure results in extremely poor self-repair capabilities after injury. Mild cartilage damage, if not addressed promptly, can easily progress to osteoarthritis, while severe injuries can lead to joint dysfunction, significantly impacting patients' quality of life. With the increasing aging population and the rising incidence of sports injuries and trauma, cartilage repair has become a pressing clinical challenge in orthopedics and sports medicine.

[0003] Collagen is the most abundant structural protein in the human body, with type II collagen (Col II) being a core component of the extracellular matrix of chondrocytes, accounting for more than 50% of the dry weight of cartilage. It provides attachment sites for chondrocytes by forming a three-dimensional fibrous network structure, while simultaneously regulating cell proliferation, differentiation, and matrix synthesis, making it a key substance for maintaining the morphology and function of cartilage tissue. Therefore, repair materials based on type II collagen are considered an ideal choice for cartilage damage repair, with biocompatibility and functional specificity far exceeding those of traditional synthetic materials.

[0004] Currently, type II collagen-related repair materials used in clinical practice and research are mainly divided into two categories: naturally extracted and recombinant expression-based. However, both have significant technical shortcomings. Naturally extracted type II collagen is mainly derived from the cartilage tissue of animals such as cattle, pigs, and chickens. Although it possesses certain biological activity, it faces three core problems: First, there is the risk of immunogenicity. The amino acid sequence difference between foreign proteins and human type II collagen can easily trigger an immune rejection reaction, leading to repair failure or inflammatory response. Second, there are safety concerns. Animal-derived materials may carry pathogens such as viruses and bacteria, posing a risk of cross-infection. Third, there is the instability in quality. The collagen structure is easily damaged during the natural extraction process, and differences in raw material sources and extraction processes lead to large fluctuations in activity and purity between batches, making it difficult to meet the requirements of standardized clinical applications.

[0005] However, studies have shown that full-length and high-dose type II collagen in animals can induce osteoarthritis (Zhang GB, Jia ZJ, Zhang MH, Li YJ, Tian LY, Cheng J. Flaccidoside II ameliorates collagen-induced arthritis in mice. Eur J Pharmacol. 2020 Aug 5;880:173155). Currently, it is being studied as a method for establishing some animal models of osteoarthritis. However, as a major component of the extracellular matrix of chondrocytes, type II collagen plays an important and crucial role in cartilage repair, but research on the specific active areas of cartilage repair is still lacking.

[0006] The development of recombinant expression technology has provided a solution to the shortcomings of natural collagen, but existing recombinant type II collagen has not yet overcome key technological bottlenecks. Early recombinant products were mostly based on prokaryotic expression systems (such as *E. coli*), which, while enabling large-scale production, lacked proper glycosylation modification and triple-helix folding, resulting in significantly lower bioactivity than natural proteins and an inability to effectively mimic the function of human cartilage matrix. Recombinant proteins produced by some eukaryotic expression systems (such as yeast and insect cells), while structurally closer to the natural type, suffer from insufficient humanization, low expression levels, and high production costs, hindering industrialization. Furthermore, the application of existing recombinant collagen in cartilage repair faces challenges such as poor persistence of bioactivity, unclear regulatory mechanisms of interaction with chondrocytes, and easy degradation of repaired tissues, limiting its clinical efficacy.

[0007] Besides collagen-based materials, existing cartilage repair techniques include autologous cartilage transplantation, allogeneic cartilage transplantation, implantation of synthetic polymer materials, and stem cell therapy. However, each has its own limitations: autologous cartilage transplantation suffers from a shortage of donors and can cause secondary damage; allogeneic transplantation carries the risk of immune rejection and disease transmission; synthetic polymer materials (such as polylactic acid and hydroxyapatite) have limited biocompatibility, lack cell regulation functions, and the repaired tissue is mostly fibrous scar tissue, making it difficult to restore the normal mechanical properties of cartilage; although stem cell therapy has differentiation potential, it suffers from problems such as uncontrollable differentiation efficiency, ethical controversies, and high costs, and has not yet achieved widespread clinical application.

[0008] In summary, there is an urgent clinical need for a cartilage repair material that is highly biocompatible, low in immunogenicity, structurally and functionally close to natural human type II collagen, and can be produced on a large scale and at low cost. Recombinant humanized type II collagen, through optimized gene sequence design and combined with a highly efficient eukaryotic expression system, can overcome the immunogenicity, safety, and activity deficiencies of existing materials, providing a novel solution for cartilage damage repair and possessing significant clinical value and industrialization prospects. Summary of the Invention

[0009] To address the aforementioned issues, this invention aims to provide a recombinant humanized type II collagen and its application in cartilage repair; the recombinant humanized type II collagen of this invention has good biological activity and can be used for cartilage repair, etc.

[0010] In one aspect, the present invention provides recombinant humanized type II collagen, characterized in that it comprises n repeating units, said repeating units being directly or connected by a linker, said repeating units comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or a variant thereof.

[0011] In some embodiments, the variant is (1) an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO:1 or SEQ ID NO:2; or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1 or SEQ ID NO:2.

[0012] In some implementations, n is an integer from 1 to 20.

[0013] In some implementations, n is 4 or 16.

[0014] In some implementations, the repeating units are directly connected to each other.

[0015] In some implementations, the repeating units are connected by a connector.

[0016] In some embodiments, the collagen comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence obtained by substituting, deleting, or inserting one or more amino acids based on SEQ ID NO:3 or SEQ ID NO:4.

[0017] In one aspect, the present invention provides a fusion protein comprising the recombinant humanized type II collagen described herein.

[0018] In this invention, the fusion protein further includes purification tags and / or precursors. The purification tags include, but are not limited to, His tags, GST tags, MBP tags, SUMO tags, or NusA tags.

[0019] In one aspect, the present invention provides a polynucleotide encoding the recombinant humanized type II collagen or the fusion protein described herein.

[0020] In some embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6, or a degenerate sequence thereof.

[0021] In some embodiments, the polynucleotide is codon-optimized for the host cell in which it is expressed.

[0022] In some implementations, the polynucleotide encoding recombinant humanized type II collagen can be operatively linked to expression control elements, such as promoters, terminators, and / or enhancers, to form a nucleic acid or expression cassette.

[0023] In some embodiments, the polynucleotide encoding recombinant humanized type II collagen may also include nucleotides encoding purification tags, such as His tags, GST tags, MBP tags, SUMO tags, or NusA tags, or nucleotides encoding leader sequences to facilitate peptide purification or secretion.

[0024] In one aspect, the present invention provides a vector comprising the polynucleotides described herein.

[0025] In some embodiments, the carrier is an expression carrier.

[0026] In some embodiments, the vector includes a control element operatively linked to a polynucleotide.

[0027] In some embodiments, the control element is selected from promoters, terminators, and / or enhancers.

[0028] In one aspect, the present invention provides a host cell comprising the polynucleotides described herein, or the vectors described herein.

[0029] In one aspect, the present invention provides a method for producing the recombinant humanized type II collagen or the fusion protein described herein, comprising the following steps:

[0030] (1) The host cells described herein are cultured under induction conditions to express the recombinant protein;

[0031] (2) Harvest host cells and / or culture medium containing recombinant humanized type II collagen or fusion protein;

[0032] (3) Purify the recombinant humanized type II collagen or fusion protein described in this article.

[0033] In one aspect, the present invention provides compositions described herein comprising one or more of the recombinant humanized type II collagen, fusion protein, polynucleotide, vector, and host cell described herein.

[0034] In some embodiments, the composition is one or more of the following: cartilage tissue repair materials, bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, and pharmaceutical excipients.

[0035] In some embodiments, the composition is a surface composition, an injectable composition, or an oral composition.

[0036] In this invention, the composition may be administered by means including but not limited to injection, topical application, or oral administration.

[0037] In one aspect, the present invention provides the use of the recombinant humanized type II collagen and / or compositions described herein in the preparation of cartilage regeneration and / or repair products.

[0038] In some embodiments, the product is a product having at least one of the following functions:

[0039] (a) Products that promote cell adhesion;

[0040] (b) Products that promote cell migration;

[0041] (c) Products that promote chondrocyte proliferation;

[0042] (d) A product that inhibits the expression of inflammatory factors; preferably, the inflammatory factors are selected from one or more of TNF-α, IL-6, IL-12, and IL-1β.

[0043] At least the following beneficial effects of the present invention:

[0044] (1) This invention provides a novel core functional region of recombinant humanized type II collagen, and successfully constructs a recombinant humanized type II collagen with good biological activity based on the novel core functional region.

[0045] (2) The amino acid composition of the recombinant humanized type II collagen of the present invention is 100% identical to the corresponding part of the amino acid sequence of natural human type II collagen, and will not produce immune rejection or allergic reaction when applied to the human body.

[0046] (3) The recombinant humanized type II collagen of the present invention can not only effectively promote the adhesion and migration of chondrocytes, but also significantly inhibit the expression of inflammatory factors, providing a fully functional biomimetic microenvironment for cartilage damage repair.

[0047] (4) The preparation method of the present invention is simple and can produce high-yield recombinant humanized type II collagen on a large scale. Attached Figure Description

[0048] Figure 1 SDS-PAGE electrophoresis images of recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2;

[0049] Figure 2 Figure showing the results of detecting the adhesion activity of recombinant humanized type II collagen on C28 / I2 chondrocytes;

[0050] Figure 3 Figure showing the effect of recombinant humanized type II collagen rhCOL2-1 on the viability of C28 / I2 chondrocytes;

[0051] Figure 4 Figure showing the effect of recombinant humanized type II collagen rhCOL2-2 on the viability of C28 / I2 chondrocytes;

[0052] Figure 5 Microscopic images of the effect of recombinant humanized type II collagen on scratch healing of NIH / 3T3 cells;

[0053] Figure 6 The figure shows the detection results of the inhibitory effect of recombinant humanized type II collagen on the expression of inflammatory factors in LPS-induced THP-1 cells. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0055] Terminology Definition

[0056] As used in this article, "recombinant collagen" is a new type of biomaterial that uses cutting-edge structural biology, genetic engineering and other technologies to screen and prepare a gene encoding the functional region of human collagen specific type as a template, and obtains an amino acid sequence that is the same as or similar to that of human collagen.

[0057] As used herein, “recombinant humanized type II collagen” refers to a recombinant protein consisting of or substantially consisting of sequences derived from human type II collagen. In this context, recombinant humanized type II collagen may consist of or substantially consist of fragments or multiple repeats of fragments derived from human type II collagen.

[0058] As used herein, “one or more” can be any suitable integer. In the case of collagen mutations (e.g., substitution, deletion, insertion, or addition), “one or more” is a number readily determined by those skilled in the art, such as 1-90 and any integers and ranges therebetween, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, etc.

[0059] As used herein, the term “expression” includes any step involved in peptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0060] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may contain the nucleic acids of this invention for introduction into cells for expression. Vectors may contain expression control elements operatively linked to the nucleic acids, such as promoters, terminators, and / or enhancers.

[0061] As used herein, the term "expression vector" refers to a straight or circular DNA molecule containing a polynucleotide encoding a polypeptide and operatively linked to a control sequence provided for its expression. In this paper, the expression vector is an *E. coli* expression vector.

[0062] As used herein, the term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding region of the polynucleotide encoding the polypeptide.

[0063] As used herein, the term "host cell" refers to a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques. Host cells can be eukaryotic or prokaryotic cells. For example, eukaryotic cells include yeast cells, animal cells, and / or insect cells. Prokaryotic cells can be E. coli cells.

[0064] In this invention, "cartilage tissue repair material" refers to a functional material based on biocompatible materials that can simulate or supplement the structure of the natural cartilage matrix, providing an adhesion and growth interface for chondrocytes, promoting cell migration, inhibiting local inflammatory responses, and supporting cartilage tissue regeneration. The recombinant humanized type II collagen described in this paper, with its excellent cell adhesion, ability to promote cell migration, and significant anti-inflammatory activity, can be used as a core active ingredient to construct such materials. These materials can be used through injection filling, implantable scaffolds, or bio-inks to promote the repair and functional reconstruction of cartilage defects.

[0065] In this invention, "biological dressing" is a medical material with a specific structure (such as membrane, sponge, gel, etc.) formed by cross-linking or processing biological materials (such as collagen, hyaluronic acid, etc.) through physical or chemical methods. It is used to cover and protect skin wounds and promote wound healing and tissue regeneration.

[0066] In this invention, "bionic materials" refers to materials developed by imitating various characteristics or properties of living organisms. Generally, artificial materials designed and manufactured in accordance with the operational patterns of living systems and the structural principles of biological materials are called biomimetic materials. Human biomimetic materials are artificial materials designed and manufactured in accordance with the operational patterns of human living systems and the structural principles of biological materials. The collagen in this invention has the effect of promoting cell adhesion, and therefore can be used in human biomimetic materials.

[0067] In this invention, "cosmetic materials" refers to biocompatible medical materials used in plastic surgery or non-surgical cosmetic treatments for filling, shaping, repairing, or improving appearance, such as soft tissue fillers, prosthetic implants, and absorbable sutures. Those skilled in the art know that collagen can be used in cosmetic procedures.

[0068] In this invention, "organoid culture material" refers to materials used for culturing organoids. Its function is to mimic the extracellular microenvironment in vivo, providing essential structural support, biochemical signal guidance, and nutrient exchange interfaces for organoid formation and development, thereby effectively promoting cell self-assembly, proliferation, differentiation, and ultimately functional expression. The collagen described in this invention has the effect of promoting cell adhesion and therefore can be used for organoid culture.

[0069] In this invention, "cardiovascular stent material" refers to the material used in the fabrication of cardiovascular stents. Such materials must possess good biocompatibility, mechanical support, and blood compatibility, and be able to remain stable in vivo for a long period or degrade in a controlled manner as expected.

[0070] In this invention, "coating material" refers to a substance that can be coated onto the surface of medical devices, implants or other materials to improve their biocompatibility, lubricity, antibacterial properties, anticoagulation, conductivity or drug sustained-release function, etc.

[0071] In this invention, "tissue injection filler material" refers to a biomaterial that can be injected into the human body and used as a structural support and functional repair matrix. These materials can be implanted into human soft tissue via minimally invasive injection to achieve volume replenishment, contour modification, functional repair, or tissue regeneration. Examples include injectable medical materials such as collagen, hyaluronic acid, and polylactic acid microspheres.

[0072] In this invention, "ophthalmic materials" refers to medical materials used for the prevention, diagnosis, or treatment of eye diseases and injuries, including artificial lenses, corneal transplant materials, intraocular tamponade, contact lenses, lacrimal plugs, and ophthalmic sustained-release drug carriers.

[0073] In this invention, "gynecological and obstetric biomaterials" refers to medical materials used for the prevention, diagnosis or treatment of female reproductive system diseases, damage or repair of supporting tissues, such as pelvic floor repair mesh, endometrial repair scaffolds, anti-adhesion barriers, obstetric hemostatic materials, etc.

[0074] In this invention, "neural repair and regeneration materials" refers to biomaterials that can regenerate damaged nerve tissue or promote functional recovery, such as nerve conduits, hydrogel scaffolds, electroactive matrices, decellularized nerve grafts, etc.

[0075] In this invention, "liver tissue materials and vascular repair and regeneration materials" refers to biomaterials used to construct artificial liver tissue models, repair liver damage, promote vascular network formation, or for vascular regeneration engineering, such as liver tissue engineering scaffolds, vascular grafts, and angiogenesis-promoting bio-inks.

[0076] In this invention, "3D printed artificial organ biomaterial" refers to the material used as the main component of "bio-ink" in 3D bioprinting to manufacture artificial tissue or organ prototypes with complex three-dimensional anatomical structures, which usually has good printing adaptability, cell compatibility and structural stability.

[0077] In this invention, "cosmetic raw materials" refers to active ingredients or functional matrix materials used in cosmetic formulations to exert specific effects (such as moisturizing, repairing, anti-oxidation, whitening, soothing, etc.), such as collagen, peptides, plant extracts, hyaluronic acid, etc.

[0078] In this invention, "pharmaceutical excipients" refer to substances contained in a pharmaceutical preparation other than the active pharmaceutical ingredient, which play roles such as excipient, stabilizer, solubilizer, sustained release, taste masker, lubricant, or improve drug delivery performance. They should meet pharmaceutical standards and not affect the stability and efficacy of the active pharmaceutical ingredient.

[0079] As used herein, the term "cartilage regeneration" refers to the formation, replacement, integration, and functional restoration of new cartilage tissue through biological processes following damage, defects, or degeneration of cartilage tissue. In this invention, the recombinant humanized type II collagen provides a biomimetic and functional microenvironment for cartilage regeneration by promoting the adhesion, migration, and proliferation of key cells and inhibiting harmful inflammation, thereby guiding and accelerating this regeneration process.

[0080] As used herein, the term "cartilage repair" refers to the structural and / or functional repair and improvement of damaged cartilage tissue. The recombinant humanized type II collagen or its composition provided by this invention not only provides physical support (repair) as a filler material, but also actively guides cell behavior through its excellent bioactivity, promoting the formation of new tissue with normal cartilage characteristics (i.e., regeneration), thereby achieving functional repair from passive filling to active guidance.

[0081] As used herein, the term "enhancing cell adhesion" refers to the ability of cells to attach, spread, and anchor on material surfaces or tissue interfaces by providing biocompatible surfaces or specific binding sites. Specifically, in this context, the recombinant humanized type II collagen or its composition provided by this invention enables chondrocytes to adhere more effectively to their surfaces, providing a basis for their subsequent proliferation, differentiation, and other behaviors.

[0082] As used in this article, the term "facilitating cell migration" refers to the ability to stimulate or guide cells to move directionally from their original location to a specific area (such as a site of injury).

[0083] As used in this article, the term "promoting chondrocyte proliferation" refers to increasing the number of chondrocytes or increasing their division rate. This is a fundamental step in replenishing the chondrocyte pool and providing sufficient functional cells for new cartilage tissue.

[0084] As used herein, the term "inhibition of inflammatory cytokine expression" refers to downregulating or reducing the production or biological activity of signaling molecules (i.e., inflammatory cytokines) associated with inflammatory responses. In pathological states such as cartilage damage or osteoarthritis, excessive inflammatory responses accelerate cartilage destruction, inhibit chondrocyte synthesis, and hinder regeneration. In this invention, specifically, the product can reduce the expression levels of key pro-inflammatory factors (such as TNF-α, IL-6, IL-12, and IL-1β) in cells or tissues, thereby creating a local microenvironment conducive to cartilage repair.

[0085] The following embodiments are provided to illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.

[0086] Example 1: Construction of recombinant humanized type II collagen

[0087] Large-scale functional region screening of natural human type II collagen yielded the following different protein functional regions.

[0088] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN (SEQ ID NO: 1);

[0089] GNAGPAGPAGPRGEVGLPGLSGPVGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPRGIPGPVGAAGATGARGLVGEPGPAGSKGESGNKGEPGSAGPQGPPGPSGEEGKRGPNGEAGSA (SEQ ID NO: 2);

[0090] To ensure the purification and stability of recombinant humanized type II collagen, the amino acid fragments in these regions were optimized through n repetitions and direct ligation, resulting in recombinant collagen rhCOL2-1 and rhCOL2-2, with corresponding amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. If ligation is performed using a linker, the linker can be a flexible peptide such as (GGGGS). n And it does not affect protein function, n = 1-4 integers.

[0091] Amino acid sequence of rhCOL2-1:

[0092] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0093] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0094] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0095] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0096] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0097] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0098] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0099] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0100] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0101] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0102] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0103] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0104] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0105] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0106] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN

[0107] GIRGHNGLDGLKGQPGAPGVKGEPGAPGEN (SEQ ID NO:3)

[0108] Amino acid sequence of rhCOL2-2:

[0109] GNAGPAGPAGPRGEVGLPGLSGPVGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPPRGIPGPVGAAGATGARGLVGEPGPAGSKGESGNKGEPGSAGPQGPPGPSGEEGKRGPNGEAGSA

[0110] GNAGPAGPAGPRGEVGLPGLSGPVGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPPRGIPGPVGAAGATGARGLVGEPGPAGSKGESGNKGEPGSAGPQGPPGPSGEEGKRGPNGEAGSA

[0111] GNAGPAGPAGPRGEVGLPGLSGPVGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPPRGIPGPVGAAGATGARGLVGEPGPAGSKGESGNKGEPGSAGPQGPPGPSGEEGKRGPNGEAGSA

[0112] GNAGPAGPAGPRGEVGLPGLSGPVGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPRGIPGPVGAAGATGARGLVGEPGPAGSKGESGNKGEPGSAGPQGPPGPSGEEGKRGPNGEAGSA (SEQ ID NO: 4)

[0113] Codon optimization was performed using the E. coli expression system to obtain the coding nucleic acid sequences for collagen rhCOL2-1 and rhCOL2-2, as shown in SEQ ID NO:5 and SEQ ID NO:6.

[0114] rhCOL2-1 nucleic acid sequence:

[0115]

[0116]

[0117] Example 2: Preparation and expression of recombinant humanized type II collagen

[0118] 1. Experimental Methods

[0119] The target recombinant expression plasmid was constructed by cloning the gene functional region synthesized in Example 1 into the pET-28a-Trx-His expression vector (Ubibio, VT1207). The validated recombinant plasmid was then introduced into Escherichia coli BL21 (DE3) competent cells.

[0120] (1) Remove BL21 (DE3) competent cells from the ultra-low temperature freezer, place them on ice until they are partially thawed, add 2 μL of recombinant plasmid and gently mix 2-3 times;

[0121] (2) The mixture was incubated in an ice bath for 30 min, then subjected to heat shock in a 42°C water bath (hot water bath) for 45-90 s, and then quickly transferred to ice and left to stand for 2 min.

[0122] (3) Transfer to a biosafety cabinet, add 700 μL of liquid LB medium to the system, and revive and incubate at 37°C and 220 rpm for 60 min;

[0123] (4) Take 200 μL of resuscitated bacterial solution and spread it evenly on LB resistant plates containing kanamycin sulfate;

[0124] (5) Finally, place the resistant plates treated in step (4) in a 37°C constant temperature incubator for overnight culture.

[0125] Several morphologically regular single colonies were selected from the above LB resistant plates and inoculated into LB liquid medium shake flasks containing kanamycin sulfate. The culture was carried out at 37°C and 220 rpm with constant temperature shaking until the bacterial culture reached the logarithmic growth phase. Then, the shake flasks were cooled to 16-30°C, and 0.5 mM IPTG was added to induce recombinant protein expression. After induction, the bacterial culture was aliquoted into centrifuge bottles, centrifuged at 4°C and 6000 rpm for 12 min, and the bacterial cells were collected. The wet weight of the bacterial cells was recorded, and samples were taken for electrophoresis to detect the expression effect.

[0126] 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℃ and homogenized twice or sonicated to disrupt the cells. After the cell disruption was completed, the bacterial suspension was collected. The disrupted bacterial suspension was aliquoted into centrifuge bottles and centrifuged at 17000 rpm and 4℃ for 30 min. The supernatant was collected.

[0127] Recombinant humanized type II collagen was purified and enzymatically digested using Ni affinity chromatography. The specific process is as follows:

[0128] (1) Crude purity: a. Equilibrate the column: Use equilibration solution (200 mmol / L sodium chloride, 25 mmol / L Tris, 20 mmol / L imidazole) to equilibrate the column, eliminate residual impurities and stabilize the packing environment, with a flow rate of 10 mL / min.

[0129] b. Sample loading: Add the supernatant after centrifugation to the column until all liquid has flowed out, at a flow rate of 5 mL / min.

[0130] c. Washing away contaminating proteins: Add 100 mL of washing solution (200 mmol / L sodium chloride, 25 mmol / L Tris, 20 mmol / L imidazole) until the liquid is completely drained, at a flow rate of 10 mL / min.

[0131] d. Collect the target protein: Add 20 mL of elution buffer (200 mmol / L sodium chloride, 25 mmol / L Tris, 250 mmol / L imidazole), at a flow rate of 10 mL / min, collect the flow-through, and perform electrophoresis detection.

[0132] e. Clean the column with 1 mol / L imidazole working solution at a flow rate of 10 mL / min.

[0133] (2) Enzyme digestion: TEV enzyme was added at a ratio of 20:1 between the total protein and the total protease of Tobacco Etch Virus (TEV), and the protein was digested at 16°C for 2 hours. The digested protein solution was placed in a dialysis bag and dialyzed at 4°C for 2 hours, then transferred to a new dialysis buffer (20 mmol / L Tris, 20 mmol / L sodium chloride) and dialyzed overnight at 4°C.

[0134] (3) Purification: a. Equilibrate the column: Equilibrate the column with solution A (20 mmol / L Tris, 20 mmol / L sodium chloride) at a flow rate of 10 mL / min. b. Load the sample: Load the sample at a flow rate of 5 mL / min and collect the flow-through sample. The sample is taken as QFL (Quaternary Fractionation Liquid) and analyzed by electrophoresis. Store the protein at 4°C. c. Elute: Wash the column with solution B (1 mol / L sodium chloride, 20 mmol / L Tris) at a volume of 5 column volumes (CV). d. Wash the column.

[0135] 2. Results Analysis

[0136] (1) Validation of target protein expression and purification effects

[0137] Depend on Figure 1As can be seen, both rhCOL2-1 and rhCOL2-2 samples exhibited single, clear protein bands in their lanes, without obvious contaminating protein bands (such as small molecule contaminating proteins, degradation fragments, or incompletely purified host protein bands). This indicates that the preparation process of "construction of E. coli BL21 (DE3) expression vector - IPTG-induced expression - homogenization / ultrasonic disruption - nickel column crude purification - TEV enzyme digestion - dialysis - secondary purification" successfully achieved efficient expression and high-purity purification of recombinant humanized type II collagen. During the purification process, contaminating proteins were thoroughly removed, with no obvious protein degradation or polymerization, which is beneficial for the purity requirements of subsequent bioactivity testing and joint tissue engineering applications.

[0138] (2) Target protein molecular weight matching

[0139] Based on the amino acid sequence of the recombinant humanized type II collagen in Example 1, the theoretical molecular weight of rhCOL2-1 is approximately 45123.44 Da; the theoretical molecular weight of rhCOL2-2 has been determined to be 41535.40 Da. Referring to the band positions of the molecular weight standards (markers) in Figure 1, the actual measured molecular weight of the rhCOL2-1 band is approximately 45000 Da, and the actual measured molecular weight of the rhCOL2-2 band is approximately 41000 Da. Both are highly consistent with their respective theoretical molecular weights, proving that the expressed and purified proteins are the target recombinant humanized type II collagen, without any target protein mismatch or abnormal expression.

[0140] (3) Stability of preparation process and uniformity of product

[0141] Figure 1 The protein bands of rhCOL2-1 and rhCOL2-2 both exhibited good band regularity, without tailing or diffusion, indicating that the molecular structures of the two recombinant humanized type II collagens are homogeneous and there are no obvious protein denaturations or heterogeneous aggregates. At the same time, the single and clear band characteristics also reflect the good stability of the preparation process, which can reproducibly prepare recombinant humanized type II collagen with homogeneous structure and qualified purity, providing process validation basis for the stability of product quality in subsequent large-scale production and clinical applications.

[0142] In summary, this invention successfully prepared high-purity recombinant humanized type II collagen (rhCOL2-1, rhCOL2-2) with molecular weight matching the theoretical value and uniform structure by using the E. coli BL21 (DE3) expression system combined with an optimized purification process. This provides qualified protein raw materials for subsequent applications in chondrocyte adhesion experiments, chondrocyte viability detection, and joint tissue engineering. It also verifies the feasibility and reliability of the preparation method of this invention in the large-scale production of recombinant humanized type II collagen.

[0143] Efficacy Experiment Example 1: C28 / I2 Cell Adhesion Assay of Recombinant Humanized Type II Collagen

[0144] 1. Implementation Method

[0145] (1) Protein sample preparation and concentration determination

[0146] The concentrations of each protein sample were determined using ultraviolet absorption spectrometry. The samples included: positive control PC (bovine type I collagen; National Institutes for Food and Drug Control, No. 380002), negative control NC (bovine serum albumin BSA), and the recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 prepared in this invention. The absorbance of each sample was measured at 215 nm and 225 nm, and the protein concentration was calculated using the formula C(μg / mL) = 144 × (A215 − A225). Subsequently, the concentrations of all samples were adjusted to a uniform 0.5 mg / mL using PBS solution.

[0147] In the formula, C represents the protein concentration in the sample, A215 represents the absorbance of the sample at 215 nm, and A225 represents the absorbance of the sample at 225 nm. Note that detection is required when A215 < 1.5. After determining the initial protein concentration, determine the amount of PBS solution used for dilution based on the initial concentration value, and adjust the concentration of all test proteins to 0.5 mg / mL. The principle of this method is to measure the characteristic absorption of peptide bonds under far-ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues that are not colorimetric by Coomassie Brilliant Blue.

[0148] (2) Cell adhesion experiment

[0149] a. Add 100 μL of various protein solutions and blank PBS solution to a 96-well plate and let it stand at room temperature for 60 min.

[0150] b. Add 10 to each hole 5 One well-cultured human normal chondrocyte C28 / I2 cell (ATCC, L689) was incubated at 37°C for 60 min.

[0151] c. Wash each well 4 times with PBS.

[0152] d. The absorbance at OD 450nm was measured using the CCK8 assay kit (Beyotime, product catalog number C0038). The cell adhesion rate was calculated based on the values ​​from the blank control. The formula is as follows: Cell adhesion rate p = {(OD test wells - OD blank wells) / (OD positive wells - OD blank wells)} × 100%, where the test wells contain solutions containing different proteins; the blank wells contain blank PBS solution; and the positive wells contain bovine type I collagen.

[0153] Cell adhesion rate reflects collagen activity. Higher protein activity allows for a better external environment to be provided to cells in a shorter time, thus aiding cell adhesion.

[0154] 2. Experimental Results

[0155] like Figure 2 As shown, compared with bovine type I collagen (positive control PC), the recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 of this invention exhibited stronger chondrocyte adhesion-promoting abilities, far exceeding those of the negative control BSA. These results demonstrate that the recombinant humanized type II collagen provided by this invention possesses excellent cell adhesion activity and can effectively mimic the cartilage matrix microenvironment, providing important in vitro experimental evidence for its application in cartilage repair materials.

[0156] Efficacy Experiment Example 2: C28 / I2 Cell Viability Assay with Recombinant Humanized Type II Collagen

[0157] 1 Experimental Methods

[0158] 1.1 Experimental Materials

[0159] (1) Cell line: Human chondrocytes C28 / I2 (ATCC, L689);

[0160] (2) Experimental reagents: CCK-8 kit (Liji Biotechnology, AC11L054), culture medium (DMEM+10% FBS), recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 to be tested;

[0161] (3) Experimental equipment: CO2 incubator, microplate reader (detection wavelength 450 nm).

[0162] 1.2 Experimental Procedure

[0163] (1) Cell seeding: C28 / I2 cells were seeded at a rate of 5 × 10⁻⁶ cells / year. 3 Cells / well were seeded at a density of 96-well plates, with 100 μL of complete culture medium added to each well, and incubated at 37°C and 5% CO2 for 24 hours.

[0164] (2) Treatment of test samples: Set up experimental groups of recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 with different concentrations, with 3-6 replicates in each group, and continue to culture for 48-72 hours;

[0165] (3) CCK-8 detection: Add 10 μL of CCK-8 reagent to each well and incubate in the dark for 1-4 hours;

[0166] (4) Absorbance measurement: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of each well at a wavelength of 450 nm. The reference wavelength can be selected as 600-650 nm.

[0167] 1.3 Data Analysis

[0168] Calculate cell viability: Cell viability (%) = [(As sample wells - Ab blank wells) / (Ac control wells - Ab blank wells)] × 100;

[0169] In the formula:

[0170] As: Absorbance of experimental / sample wells (including cells, culture medium, CCK8 solution, and protein solution);

[0171] Ac: Absorbance of control wells (including cells, culture medium, and CCK8 solution, but excluding protein solution);

[0172] Ab: Absorbance of blank wells (including culture medium and CCK8 solution, excluding cells and protein solution).

[0173] 2. Experimental Results

[0174] like Figure 3 and Figure 4 As shown, the cell viability of all concentration treatment groups remained close to 100%, and there was no concentration-dependent decrease in cell viability from the highest concentration of 333 μg / mL to the lowest concentration of 0.05 μg / mL.

[0175] This indicates that the recombinant humanized type II collagen did not inhibit cell viability or exhibit cytotoxicity at high concentrations. The experimental results confirm that the recombinant humanized type II collagen provided by this invention has good cell compatibility, laying a biosafety foundation for its application in biomedical fields such as tissue engineering and articular cartilage repair.

[0176] Efficacy Test Example 3: Recombinant Humanized Type II Collagen Cell Scratch Assay

[0177] 1 Experimental Methods

[0178] 1.1 Experimental Materials

[0179] (1) Cell line: NIH / 3T3 mouse fibroblasts (purchased from the Cell Bank of the Chinese Academy of Sciences);

[0180] (2) Experimental reagents: DMEM high glucose medium (Gibco), fetal bovine serum (FBS, Pronosel, 164210-50), phosphate buffer (PBS, pH 7.4), recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 (prepared in this invention), positive control PC chicken sternal cartilage type II collagen (C9301, Sigma);

[0181] (3) Experimental instruments: CO2 constant temperature incubator (Thermo), inverted microscope (Olympus), biosafety cabinet (Thermo), ImageJ image analysis software, and microplate reader.

[0182] 1.2 Experimental Methods

[0183] (1) Cell culture: NIH / 3T3 cells were seeded in DMEM high glucose medium containing 10% FBS and cultured in a 37℃, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged and used for experiments.

[0184] (2) Preparation of scratch model: NIH / 3T3 cells were prepared at a concentration of 5×10⁻⁶. 5 Cells were seeded at a density of 10 μL per well in 24-well plates and cultured for 24 h until the cells were completely confluent (confluence ≥ 95%). Using a sterile 10 μL pipette tip perpendicular to the bottom of the plate, a uniform scratch was made along the center of each well (ensuring consistent scratch width). The plate was then gently rinsed three times with PBS buffer to remove any floating cells and cell debris caused by the scratch.

[0185] (3) Group the processed orifice plates:

[0186] Experimental group: DMEM high glucose medium containing 10% FBS and 100 μg / mL recombinant humanized type II collagen was added;

[0187] Positive control PC: Chicken type II collagen (concentration controlled to be consistent with that of the experimental group);

[0188] Negative control (NC): DMEM high-glucose medium containing 10% FBS;

[0189] Each group was set up with 3 replicates and placed in an incubator at 37°C and 5% CO2 for further incubation.

[0190] (4) Observation and detection: At 0h, 24h and 48h of culture, images of the scratch area were taken under the same field of view using an inverted microscope (magnification 100×); the average width of the scratch at each time point was measured using ImageJ software, and the scratch healing rate was calculated.

[0191] Scratch healing rate (%) = (0h scratch width - scratch width at each time point) / 0h scratch width × 100%.

[0192] 1.3 Statistical Analysis

[0193] Experimental data are expressed as mean ± standard deviation (x ± s). Independent samples t-tests were performed using GraphPad software. P < 0.05 was considered statistically significant.

[0194] 2. Experimental Results

[0195] This study evaluated the promoting effect of recombinant humanized type II collagen on cell migration using the NIH / 3T3 cell scratch assay. The results of one sample are shown below. Figure 5 As shown in the photos, the average scratch healing rate is shown in Table 1. At the same concentration (100 μg / mL), the scratch healing rates of the rhCOL2-1 and rhCOL2-2 treatment groups at each time point (6h, 12h, 24h) were significantly higher than those of the positive control group (chicken type II collagen) and the negative control group, indicating that both recombinant humanized type II collagen can effectively promote the migration of fibroblasts.

[0196] Specifically, within 24 hours, the scratch healing rate of the rhCOL2-1 and rhCOL2-2 treatment groups reached approximately 80%, significantly better than the positive and negative controls (approximately 50-60%). This result indicates that the recombinant humanized type II collagen provided by this invention has excellent cell migration promotion capabilities, and cell migration is a crucial step in tissue repair. This provides cellular-level functional evidence for the application of rhCOL2-1 and rhCOL2-2 in cartilage repair.

[0197] Table 1 Scratch healing rate

[0198]

[0199] Example 4: Validation Experiment of Inflammatory Factors from Recombinant Humanized Type II Collagen

[0200] 1 Experimental Methods

[0201] To further investigate the biocompatibility of recombinant humanized type II collagen and to further verify the inhibitory effect of recombinant humanized type II collagen on lipopolysaccharide (LPS)-induced inflammatory response in THP-1 cells, the relative mRNA expression levels of TNF-α, IL-6, and IL-12 were specifically evaluated to clarify its anti-inflammatory activity.

[0202] 1.1 Experimental Materials

[0203] (1) Cells: Human monocytic leukemia cells (THP-1);

[0204] (2) Experimental reagents: recombinant humanized type II collagen, lipopolysaccharide (LPS, Sigma-Aldrich L5293), RPMI 1640 medium, fetal bovine serum (FBS), TRIzol lysis buffer (Invitrogen, 10296028), HiScript II One Step qRT-PCR SYBR Green Kit (Novizan, Q221), specific primers for human TNF-α, IL-6, and IL-12 genes (synthesized by BGI, sequences are shown in Table 2), internal reference gene GAPDH primers (synthesized by BGI, sequences are shown in Table 2), RNase-free ddH2O;

[0205] (3) Experimental instruments: CO2 cell culture incubator, centrifuge, ultra-low temperature freezer (-80℃), RT-qPCR instrument.

[0206] (4) Experimental groups: LPS group (RPMI 1640+LPS), rhCOL2-1 group (10 μg of rhCOL2-1 + RPMI1640 +LPS), rhCOL2-2 group (10 μg of rhCOL2-2 + RPMI 1640+LPS).

[0207] 1.2 Experimental Procedure

[0208] (1) Cell seeding and treatment

[0209] a. 24 hours in advance, prepare THP-1 cells at a rate of 5 × 10⁻⁶. 5 Seeds were placed at a density of 50 cells / well into 12-well cell culture plates, and RPMI 1640 medium containing 10% FBS was added to each well. The plates were then incubated in a 37°C, 5% CO2 cell culture incubator for routine culture.

[0210] b. 24 hours after inoculation, recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 were added to the wells of the pre-seeded cells in the rhCOL2-1 and rhCOL2-2 groups to a final concentration of 10 μg / well, and pretreated for 1 hour. Then, the same concentration of LPS was added to all groups to a final concentration of 100 ng / mL, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0211] c. After culture, collect the cells from each well into a sterile centrifuge tube, centrifuge at 800g for 3 minutes, discard the supernatant, add 1 mL of TRIzol lysis buffer to the precipitate, lyse thoroughly, and then freeze in an ultra-low temperature freezer at -80℃ for later use.

[0212] (2) RNA extraction

[0213] Total RNA was extracted from the frozen lysis samples to ensure that the RNA purity (A260 / A280 = 1.8~2.0) and integrity met the requirements for RT-qPCR detection.

[0214] (3) RT-qPCR detection

[0215] RT-qPCR detection was performed using the SYBR dye method (HiScript II One Step qRT-PCR SYBR Green Kit), as detailed below:

[0216] Reaction system preparation: Add each reagent sequentially to the RNase-free centrifuge tube according to the proportions shown in Table 3 to prepare a 10 μL / well RT-qPCR reaction system;

[0217] Reaction program settings: Transfer the prepared reaction system to a qPCR-specific reaction plate and place it in an RT-qPCR instrument. Amplify according to the following program: ① Reverse transcription: 50℃ for 3 minutes (1 cycle); ② Pre-denaturation: 95℃ for 30 seconds (1 cycle); ③ Cyclic reaction: 95℃ for 10 seconds, 60℃ for 30 seconds (40 cycles); ④ Melting curve analysis: 95℃ for 15 seconds, 60℃ for 60 seconds, 95℃ for 15 seconds (1 cycle);

[0218] Primer sequences: The specific primers and internal reference gene primer sequences used in this experiment are shown below.

[0219] Table 2 Primer sequences used in RT-qPCR detection

[0220]

[0221] Table 3 RT-qPCR reaction system

[0222]

[0223] 1.3 Data Processing and Statistical Analysis

[0224] The relative mRNA expression levels of each inflammatory factor (TNF-α, IL-6, IL-12) were calculated using the 2⁻ΔΔCt method, and the expression levels of the target gene were calibrated using GAPDH as an internal reference gene.

[0225] The experiment was conducted in three biological replicates. Data are expressed as mean ± standard deviation (x ± s). Statistical analysis was performed using t-tests or one-way ANOVA. A p-value < 0.05 was considered statistically significant. The study aimed to determine whether LPS-induced expression of inflammatory factors in THP-1 cells was significantly reduced after treatment with recombinant humanized type II collagen.

[0226] 2. Experimental Results

[0227] The inhibitory effects of recombinant humanized type II collagen rhCOL2-1 and rhCOL2-2 on lipopolysaccharide (LPS)-induced inflammatory responses in THP-1 cells were detected by RT-qPCR. Figure 6 As shown in Table 4, compared with the LPS treatment group alone, the mRNA expression levels of inflammatory factors TNF-α, IL-6 and IL-12 were significantly reduced after pretreatment with rhCOL2-1 or rhCOL2-2.

[0228] The LPS group was set to 1.0 for data analysis. The results are shown in Table 4: treatment with rhCOL2-1 and rhCOL2-2 significantly downregulated the mRNA expression levels of TNF-α, IL-6, and IL-12, showing a significant difference from the control group.

[0229] The results show that the recombinant humanized type II collagen provided by this invention can effectively inhibit the expression of inflammatory factors and has significant in vitro anti-inflammatory activity, providing experimental evidence for its application in the repair of inflammation-related cartilage damage.

[0230] Table 4. mRNA expression of inflammatory factors

[0231]

[0232] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Recombinant humanized type II collagen, characterized in that, It comprises n repeating units, which are connected directly or via a linker, and each repeating unit comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or a variant thereof, wherein the variant is (1) an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO:1 or SEQ ID NO:2; or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1 or SEQ ID NO:2; Preferably, n is an integer from 1 to 20; Preferably, n is 4 or 16; Preferably, the repeating units are directly connected to each other; Preferably, the collagen comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3 or SEQ ID NO:4, or an amino acid sequence obtained by substituting, deleting, or inserting one or more amino acids based on SEQ ID NO:3 or SEQ ID NO:

4.

2. A fusion protein comprising the recombinant humanized type II collagen as described in claim 1.

3. A polynucleotide encoding the recombinant humanized type II collagen of claim 1 or the fusion protein of claim 2; preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6 or a degenerate sequence thereof.

4. A vector comprising the polynucleotide of claim 3; preferably, the vector is an expression vector; preferably, the vector comprises a control element operatively linked to the polynucleotide; preferably, the control element is selected from promoters, terminators and / or enhancers.

5. A host cell comprising the polynucleotide of claim 3 or the vector of claim 4; preferably, the host cell is a bacterium, fungus or animal cell; preferably, the bacterium includes Escherichia coli.

6. A method for producing the recombinant humanized type II collagen according to claim 1 or the fusion protein according to claim 2, characterized in that, Includes the following steps: (1) The host cells of claim 5 are cultured under induction conditions to express the recombinant protein; (2) Harvest host cells and / or culture medium containing recombinant humanized type II collagen or fusion protein; (3) Purify the recombinant humanized type II collagen or fusion protein.

7. The composition, characterized in that, It comprises one or more of the following: the recombinant humanized type II collagen of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the vector of claim 4, and the host cell of claim 5; Preferably, the composition is one or more of the following: cartilage tissue repair materials, bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, and pharmaceutical excipients; preferably, the composition is a surface composition, an injectable composition, or an oral composition.

8. The use of the recombinant humanized type II collagen of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the carrier of claim 4, and the host cell of claim 5 in the preparation of one or more of the following: cartilage tissue repair materials, bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, and pharmaceutical excipients.

9. Use of the recombinant humanized type II collagen of claim 1 and / or the composition of claim 7 in the preparation of cartilage regeneration and / or repair products.

10. The use according to claim 9, characterized in that, The product is a product that has at least one of the following functions: (a) Products that promote cell adhesion; (b) Products that promote cell migration; (c) Products that promote chondrocyte proliferation; (d) A product that inhibits the expression of inflammatory factors; preferably, the inflammatory factors are selected from one or more of TNF-α, IL-6, IL-12, and IL-1β.

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