A polypeptide, recombinant humanized collagen type XIX and application thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly discloses a polypeptide, a recombinant humanized collagen of type XIX and application thereof. The amino acid sequence of the polypeptide comprises n repeating units; the repeating unit comprises any one of the following amino acid sequences or a variant sequence thereof: (1) an amino acid sequence shown in any one of SEQ ID NO. 1-2; (2) an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO. 1-2; (3) a variant sequence obtained by mutating one or more amino acid residues in the amino acid sequence shown in any one of SEQ ID NO. 1-2; wherein n is an integer greater than or equal to 1. The polypeptide has a collagen triple helix structure, has good cell adhesion activity, can perform the function of human collagen, has no immune rejection and allergic reaction, and can be used in various fields such as cartilage repair, biological dressing and tissue filling.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a polypeptide, recombinant XIX-type humanized collagen, and their applications. Background Technology

[0002] Collagen is a type of protein widely distributed in human connective tissues and is the most abundant protein in the human body, accounting for 25% to 35% of total protein. At least 28 collagen subtypes have been discovered in the human body, each located in different tissues and organs.

[0003] However, natural collagen is present in low concentrations in organisms, its extraction process is complex, and animal-derived immune responses are a significant limitation in its applications. With the development of genetic engineering technology, recombinant collagen can be constructed by selecting short amino acid sequences from natural human collagen. This recombinant collagen exhibits advantages such as low immunogenicity, high bioactivity, and good stability. As the collagen industry continues to grow, obtaining collagen through biosynthetic pathways, especially humanized collagen, is becoming increasingly mature. Recombinant humanized collagen refers to the full-length or partial amino acid sequence functional region encoded by a specific type of human collagen gene prepared using DNA recombination technology, or a combination of functional regions containing human collagen functions.

[0004] Type XIX collagen is a type of collagen found in the human body. While research on it is relatively limited, it has been found to have the following effects and functions: 1. Maintaining the structure and function of tissues and organs: Type XIX collagen is a component of the basement membrane and plays an important role in tissues and organs such as the skin, eyes, and kidneys. It helps maintain the integrity and stability of the basement membrane, providing support and fixation for cells, thereby ensuring the normal structure and function of tissues and organs. For example, in the skin, Type XIX collagen, along with other collagens, participates in the formation of the skin's scaffold structure, helping to maintain the skin's elasticity and resilience. 2. Participating in cell signal transduction: Type XIX collagen can interact with receptors on the cell surface, participating in cell signal transduction processes. Type XIX collagen can regulate cell proliferation, differentiation, migration, and survival, which is of great significance for tissue development, repair, and regeneration. For example, it affects cell behavior during wound healing, promoting the proliferation and migration of cells around the wound and accelerating wound healing. 3. Promoting angiogenesis: Studies have shown that Type XIX collagen plays a certain role in angiogenesis. It can regulate the behavior of vascular endothelial cells, promote their proliferation and migration, thereby contributing to the formation of new blood vessels. This is crucial for tissue nutrient supply and the removal of metabolic waste, and plays an important role in embryonic development, tissue repair, and tumor growth.

[0005] Although type XIX collagen possesses these potential benefits and functions, research on it is still ongoing, and many specific mechanisms and functions remain unclear. Currently, research on recombinant type XIX collagen is even scarcer, and its application in areas such as cell adhesion and proliferation is limited by issues such as unmodified post-translational processes, failure to form a higher-order triple helix conformation, low biological activity, and poor stability due to the lack of helical structure.

[0006] Therefore, it is necessary to study novel XIX-type collagen. Summary of the Invention

[0007] One of the objectives of this invention is to provide a protein that has good cell adhesion activity, is non-cytotoxic, and has a typical triple helix structure.

[0008] The present invention provides a polypeptide, wherein the amino acid sequence of the polypeptide comprises n repeating units;

[0009] The repeating unit comprises any one of the following amino acid sequences (1)-(3) or a variant thereof:

[0010] (1) The amino acid sequence shown in any one of SEQ ID NO. 1-2;

[0011] (2) An amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO. 1-2;

[0012] (3) A variant sequence obtained by mutating one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NO. 1-2;

[0013] Where n ≥ 1 and is an integer.

[0014] This invention utilizes synthetic biology and structural biology techniques to develop a polypeptide with a triple helix structure of human type XIX collagen and capable of performing the functions of human collagen. It exhibits good cell adhesion activity, is non-cytotoxic, and is suitable for use in various fields such as biological dressings, tissue filling, and cartilage regeneration.

[0015] In the polypeptide of the present invention, n is an integer between 1 and 20, preferably an integer between 4 and 12; more preferably 5, 6, 7 or 8;

[0016] Preferably, when n≥2, the repeating units are directly connected;

[0017] Preferably, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO. 3-4.

[0018] 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.

[0019] The present invention also provides a recombinant XIX type humanized collagen, wherein the recombinant XIX type humanized collagen comprises the above-mentioned polypeptide;

[0020] Preferably, the recombinant XIX type humanized collagen has cell adhesion activity.

[0021] In addition to the aforementioned polypeptides, the recombinant XIX-type humanized collagen of the present invention may also contain other polypeptide fragments from the XIX-type humanized collagen.

[0022] The present invention also provides a polynucleotide, wherein the polynucleotide encodes the above-mentioned polypeptide or fusion protein or recombinant XIX type humanized collagen;

[0023] Preferably, the polynucleotide comprises the nucleotide sequence shown in any one of SEQ ID NO. 5-6 or a degenerate sequence thereof.

[0024] The polynucleotide of the present invention 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.

[0025] The present invention also provides a biomaterial comprising the above-mentioned polynucleotides;

[0026] The biomaterial is an expression cassette, vector, or host cell;

[0027] 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.

[0028] 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.

[0029] The present invention also provides a method for producing the above-mentioned polypeptide or fusion protein or recombinant XIX type humanized 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 XIX type humanized collagen, and isolating the polypeptide or the fusion protein or the recombinant XIX type humanized collagen therefrom.

[0030] The polypeptides of the present invention can be prepared using methods known in the art.

[0031] In a preferred embodiment, the method for preparing the polypeptide of the present invention includes:

[0032] (1) Culture the above-mentioned host cells containing DNA molecules encoding the polypeptides of the present invention under suitable culture conditions;

[0033] (2) Harvesting host cells and / or culture medium containing the polypeptides of the present invention; and optionally...

[0034] (3) Purify the protein.

[0035] The present invention also provides a composition comprising one or more selected from the above-mentioned polypeptides, fusion proteins, recombinant XIX type humanized collagen, polynucleotides, and biomaterials.

[0036] The compositions of the present invention are 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;

[0037] Preferably, the composition is a topical composition, an injectable composition, or an oral composition;

[0038] Preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge, or fiber.

[0039] This invention also provides the application of the above-mentioned polypeptides, fusion proteins, recombinant XIX 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.

[0040] The present invention also provides the use of the above-mentioned polypeptides, fusion proteins, recombinant XIX type humanized collagen, polynucleotides, biomaterials or compositions in the preparation of products for any one or more of the following: cell adhesion, tissue repair, tissue filling or volume enhancement.

[0041] The present invention also provides the application of the above-mentioned polypeptides, fusion proteins or recombinant XIX type humanized collagen in any one or more of the following: cell adhesion, tissue repair, tissue filling or volume enhancement, and cosmetic surgery, wherein the application is for non-disease diagnosis or treatment purposes.

[0042] The present invention also provides the use of the above-mentioned polypeptides, fusion proteins, recombinant type XIX humanized collagen, polynucleotides, biomaterials or compositions in the preparation of medicaments for the prevention and / or treatment of diseases or conditions related to type XIX collagen deficiency or for the supplementation of type XIX collagen.

[0043] The present invention also provides a method for promoting cell adhesion, the method comprising the step of contacting the above-mentioned polypeptide, fusion protein, recombinant XIX type humanized collagen or composition with cells; the cells are preferably animal cells; more preferably mammalian cells; more preferably human cells; preferably, the promotion of cell adhesion is performed in vivo / in vitro, and the method is for non-disease diagnosis or treatment purposes.

[0044] Terminology Definition

[0045] 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.

[0046] As used herein, “recombinant type XIX humanized collagen” refers to a recombinant protein consisting of or substantially consisting of sequences derived from human type XIX collagen. In this context, recombinant type XIX humanized collagen may consist of or substantially consist of fragments or multiple repeats of fragments derived from human type XIX collagen.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] As used herein, the term “recombinant expression vector” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain nucleic acid segments in a manner not normally found in nature, or is synthetic and contains one or more control sequences.

[0051] 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 different genes) 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.

[0052] 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.

[0053] The beneficial effects of this invention are at least as follows:

[0054] This invention provides a novel polypeptide with a three-dimensional helical conformation, good cell adhesion activity, no cytotoxicity, and injectability into the human body—recombinant XIX type humanized collagen. The human body will not produce immune rejection or allergic reactions to it. It can be used in various fields such as tissue repair, biological dressings, and tissue filling. Moreover, the preparation method is simple and can be mass-produced. Attached Figure Description

[0055] Figure 1 The results of SDS-PAGE analysis of the purified recombinant protein XIX-1 are shown.

[0056] Figure 2 The results of SDS-PAGE analysis of the purified recombinant protein XIX-2 are shown.

[0057] Figure 3 The results are shown in the circular dichroism UV-Vis analysis of recombinant protein XIX-1.

[0058] Figure 4 The results are shown in the circular dichroism UV-Vis analysis of the recombinant protein XIX-2.

[0059] Figure 5 The figure shows the cell adhesion activity assay results for recombinant proteins XIX-1 and XIX-2. This means P < 0.05. This means P < 0.001.

[0060] Figure 6 The results show the relative cell viability of recombinant proteins XIX-1 and XIX-2. Detailed Implementation

[0061] 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.

[0062] 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.

[0063] Construction and production method of recombinant XIX type humanized collagen

[0064] This invention provides a method for constructing recombinant XIX type humanized collagen, including functional region screening and strain construction, large-scale bio-fermentation, protein induction expression, purification, and optionally enzymatic digestion steps.

[0065] In some embodiments, the functional region screening and strain construction steps include: (1) large-scale functional region screening study 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.

[0066] In some embodiments, the large-scale bio-fermentation step includes: adding the screened positive Escherichia coli genetically engineered bacteria to a shake flask containing an antibiotic stock solution and culturing it in a constant-temperature shaker.

[0067] In some embodiments, the steps for inducing protein expression include: (1) cooling the cultured shake flask to 16-30°C; (2) adding IPTG stock solution to induce expression; and (3) placing the induced bacterial culture into a centrifuge bottle, centrifuging, and collecting the bacterial cells.

[0068] In some embodiments, the purification and optional enzymatic digestion steps of recombinant XIX type humanized collagen include: (1) crude purification of recombinant XIX type humanized collagen on a Ni affinity chromatography column; (2) enzymatic digestion with TEV enzyme added in a certain proportion; and (3) purification of recombinant XIX type humanized collagen on an ion exchange column.

[0069] 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.

[0070] Example 1: Construction and expression of recombinant XIX type humanized collagen (the polypeptide of this invention)

[0071] 1.1 Construction of recombinant XIX type humanized collagen

[0072] The present invention has identified the following different protein functional regions through research.

[0073] SEQ ID NO.1:

[0074] GPPGPPGPKGDPGPVGEPGAMGLPGLEGFPGVKGDRGPAGPP;

[0075] SEQ ID NO.2:

[0076] GEPGLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQ;

[0077] The present invention further optimizes the above amino acid fragments by repeating them n times and directly linking them to obtain recombinant collagen XIX-1 and collagen XIX-2, the corresponding amino acid sequences of which are shown in SEQ ID NO:3 and EQ ID NO:4, respectively.

[0078] The amino acid sequence of recombinant XIX-type humanized collagen XIX-1 (5 repeats):

[0079] GPPGPPGPKGDPGPVGEPGAMGLPGLEGFPGVKGDRGPAGPPGPPGPPGPKGDPGPVGEPGAMGLPGLEGFPGVKGDRGPAGPPGPPGPPGPKGDPGPVGEPGAMGLPGLEGFPGVKGDRGPAGPPGPPGPPGPKGDPGPVGEPGAMGLPGLEGFPGVKGDRGPAGPPGPPGPPGPKGDPGPVGEPGAMGLPGLEGFPGVKGDRGPAGPP (SEQ IDNO.3);

[0080] The amino acid sequence of recombinant XIX-type humanized collagen XIX-2 (6 repeats):

[0081] GEPGLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQGEPGLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQGEPGLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQGEP GLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQGEPGLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQGEPGLDGNPGAPGPRGPKGERGLPGVHGSPGDIGPQ (SEQID NO.4).

[0082] Codon optimization was performed using the E. coli expression system to obtain the coding nucleic acid sequences for collagen XIX-1 and collagen XIX-2, as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0083] Collagen XIX-1 nucleic acid sequence:

[0084] GGGCCCCCAGGACCGCCGGGTCCGAAAGGTGATCCGGGTCCGGTAGGTGAGCCGGGTGCGATGGGCTTGCCGGGGTTGGAGGGCTTTCCGGGCGTCAAAGGTGATCGTGGCCCGGCGGGTCCGCCAGGCCCGCCGGGTCCGCCAGGTCCGAAGGGTGACCCAGGCCCAGTGGGCGAGCCCGGTGCAATGGGTCTGCCGGGTCTGGAAGGCTTCCCGGGCGTGAAGGGCGACCGCGGTCCTGCGGGTCCGCCAGGTCCGCCGGGCCCACCGGGTCCGAAAGGCGACCCGGGTCCGGTGGGTGAACCGGGCGCTATGGGTCTGCCGGGTCTTGAGGGTTTCCCGGGCGTTAAGGGCGACCGCGGTCCGGCGGGTCCGCCGGGCCCTCCGGGTCCGCCGGGCCCGAAGGGTGACCCGGGCCCGGTTGGTGAGCCGGGAGCGATGGGTTTACCGGGCCTGGAAGGTTTCCCGGGCGTTAAAGGCGATCGTGGTCCGGCTGGCCCGCCGGGCCCGCCGGGCCCACCGGGCCCGAAAGGCGATCCGGGTCCGGTGGGTGAACCGGGCGCAATGGGTCTGCCTGGCCTGGAAGGTTTTCCGGGCGTTAAGGGCGATCGTGGTCCGGCGGGTCCACCG (SEQ ID NO: 5).

[0085] Collagen XIX-2 nucleic acid sequence:

[0086] GGAGAACCCGGGCTGGACGGTAACCCGGGCGCGCCGGGTCCGAGAGGCCCTAAAGGTGAACGCGGCCTGCCGGGTGTCCACGGCTCCCCGGGTGACATCGGCCCGCAGGGCGAGCCGGGCTTGGACGGCAACCCGGGTGCGCCAGGTCCGCGTGGTCCGAAAGGTGAGCGCGGTCTGCCGGGGGTGCATGGTTCTCCGGGTGATATTGGTCCGCAAGGTGAGCCGGGCCTTGACGGCAACCCGGGTGCCCCTGGCCCGCGTGGTCCGAAGGGTGAACGTGGTCTGCCGGGTGTTCATGGTAGCCCGGGTGATATTGGCCCACAAGGTGAGCCGGGACTGGATGGCAATCCGGGAGCACCGGGTCCGCGTGGTCCGAAGGGCGAGCGCGGCTTACCGGGCGTTCACGGTAGCCCGGGCGATATTGGTCCGCAGGGTGAACCGGGCCTGGATGGTAATCCGGGTGCTCCGGGTCCACGTGGTCCGAAAGGTGAACGTGGCCTGCCTGGCGTGCACGGCAGCCCGGGCGATATCGGTCCGCAAGGTGAGCCGGGCTTGGACGGCAACCCGGGCGCGCCAGGCCCACGCGGTCCGAAGGGTGAACGTGGCCTCCCGGGTGTTCACGGCTCGCCGGGCGACATCGGCCCACAG (SEQ ID NO: 6).

[0087] 1.2 Expression of Recombinant Humanized Collagen Type XIX

[0088] Insert the synthesized gene functional region into the pET-28a-Trx-His expression vector to obtain the corresponding recombinant expression plasmid.

[0089] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process was as follows: (1) The E. coli competent cells BL21(DE3) were taken out of the ultra-low temperature freezer and placed on ice. When they were half-thawed, 2 μl of the plasmid to be transformed was added to the E. coli competent cells BL21(DE3) and mixed slightly 2-3 times. (2) The mixture was placed on ice for 30 min, and then heat-shocked in a water bath at 42℃ for 45-90 s. After taking it out, it was placed on ice for 2 min. (3) It was transferred to a biosafety cabinet and 700 μl of liquid LB medium was added. Then it was cultured at 37℃ and 220 rpm for 60 min. (4) 200 μl of bacterial solution was evenly spread on LB plates containing kanamycin sulfate. (5) The plates were cultured in an incubator at 37℃ for 15-17 h until uniformly sized colonies grew.

[0090] Pick 5-6 single colonies from the transformed LB agar plates and place them in a shake flask containing antibiotic stock solution (ampicillin 100 mg / L). Incubate at 220 rpm and 37°C in a constant temperature shaker for a certain period of time until the colonies appear as a mist. 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, record the cell weight, and perform electrophoresis analysis.

[0091] 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.

[0092] The recombinant humanized XIX type collagen was purified and enzymatically digested. The specific process was as follows: (1) Crude purification: a. Equilibrate the column: Equilibrate the column with equilibration buffer (200mM sodium chloride, 25mM Tris, 20mM imidazole) at a flow rate of 10mL / min. b. Load the sample: Add the supernatant after centrifugation to the column until the liquid is completely discharged at a flow rate of 5mL / min. c. Wash away impurities: Add 100 mL of washing buffer (200 mM sodium chloride, 25 mM Tris, 20mM imidazole) until the liquid is completely discharged at a flow rate of 10mL / min. d. Collect the target protein: Add 20 mL of elution buffer (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole), at a flow rate of 10 mL / min, and collect the flow-through. Detect the protein concentration using UV-Vis spectrophotometry. Calculate the protein concentration using the formula (C (mg / ml) = A280 × dilution factor × extinction coefficient), and perform electrophoresis. e. Wash the column with 1 M imidazole working solution. The working solution pH is 7.5, and the flow rate is 8 mL / min. (2) Enzyme digestion: Add a certain amount of TEV enzyme according to the total protein amount and digest at 16℃. The specific enzyme addition amount, digestion time, and digestion effect are shown in Tables 1 and 2. Place the digested protein solution into a dialysis bag and dialyze at 4℃ for 2-4 h. Then transfer it to a new dialysis buffer (20 mM sodium chloride, 20 mM Tris) and dialyze overnight at 4℃. (3) Purification: a. Column equilibration: Equilibrate the column with solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 ml / min. b. Sample loading: Load the sample at a flow rate of 5 ml / min, collect the flow-through sample, and perform electrophoresis. Store the protein at 4°C. c. Elution: Wash the column with solution B (1 M sodium chloride, 20 mM Tris) for 5 CVs. d. Column washing.

[0093] Table 1 Selection of XIX-1 enzyme digestion ratio

[0094]

[0095] Table 2 Selection of XIX-2 enzyme digestion ratio

[0096]

[0097] Based on the above experimental results, for XIX-1, a suitable protein-to-TEV enzyme ratio of 15:1 was selected, and complete enzyme digestion was achieved after 2 hours; for XIX-2, a suitable protein-to-TEV enzyme ratio of 10:1 was selected, and complete enzyme digestion was achieved after 2 hours.

[0098] The two recombinant proteins obtained after purification using the preferred enzyme digestion method described above for XIX-1 and XIX-2 proteins were detected by SDS-PAGE. The electrophoresis results are shown below. Figures 1-2 .like Figures 1-2 It can be seen that collagen XIX-1 and collagen XIX-2 were successfully expressed and prepared. The theoretical molecular weight of recombinant XIX-type humanized collagen XIX-1 is 19054.51 Da, and the apparent molecular weight after purification is basically consistent with the theoretical molecular weight. The theoretical molecular weight of recombinant XIX-type humanized collagen XIX-2 is 20217.96 Da, and the apparent molecular weight after purification is basically consistent with the theoretical molecular weight. Both collagens were accurately expressed and successfully prepared.

[0099] Subsequent purification showed that the recombinant XIX-type humanized collagen XIX-1 underwent complete protease cleavage, resulting in a clear and single target band with good purity and a yield of 0.72%. The recombinant XIX-type humanized collagen XIX-2 exhibited high protein purification yield and purity, with complete protease cleavage and a yield of 1.11%. The protein yield was determined using the following method: Protein yield = Protein expression level (g) ÷ Cell weight (g) × 100%.

[0100] Example 2: Circular dichroism UV-Vis analysis of recombinant XIX type humanized collagen

[0101] Experimental methods

[0102] (1) Instrument parameter settings

[0103] Bandwidth: 1.0nm; Step: 1.0nm;

[0104] Measurement range: 190-260nm (far-UV region scanning) / 250-340nm (near-UV region scanning);

[0105] Time per point: 0.5s; Repeats: 3 times;

[0106] Cell Length: 10mm | 0.5mm; Temperature: Room temperature.

[0107] (2) Near and far ultraviolet scanning of standard samples

[0108] The scanning wavelength was set to 180-340 nm for background testing and blank buffer testing. Then, the circular dichroism near and far UV absorption of 1 mg / mL CSA standard solution in the 180-340 nm range was collected.

[0109] (3) Sample processing

[0110] The protein sample prepared in Example 1 was concentrated to a protein concentration of 1 mg / ml using a 10 KD ultrafiltration concentrator (Millibo).

[0111] (4) Far-ultraviolet scanning of samples

[0112] 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.

[0113] (5) Near-ultraviolet scanning of samples

[0114] 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

[0116] All scanned spectra were processed using the software Pro-Data Viewer to extract baselines and smooth out the images.

[0117] (7) Experimental results and analysis

[0118] The results showed that both recombinant XIX-type humanized collagen proteins exhibited positive peaks at 220-230 nm, indicating that both recombinant collagen proteins possess a triple helix structure. (See attached image for details.) Figure 3 and Figure 4 The triple helix structure of the XIX-1 protein is superior to that of XIX-2, resulting in a higher peak value.

[0119] Example 3: Detection of Adhesion Activity of Recombinant XIX Type Humanized Collagen

[0120] The method for detecting collagen activity can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0121] (1) The concentration of the protein samples to be tested was detected using ultraviolet absorption, including bovine type I collagen (China National Institutes for Food and Drug Control, No.: 380002) and recombinant collagen XIX-1 and XIX-2 prepared in Example 1 of this invention. Specifically, the ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated using the empirical formula C(μg / mL) = 144 × (A215 - A225). Note that the detection must be performed when A215 < 1.5. The principle of this method is: measuring 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 do not show color in Coomassie Brilliant Blue (reference: Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43-45.). After the protein concentration was measured, the concentration of all the proteins to be tested was adjusted to 1 mg / mL with PBS.

[0122] (2) Sample preparation: The original sample solution was used directly for the experiment; the positive control bovine type I collagen (PC) was diluted to 1 mg / ml with D-PBS for later use; the negative control was D-PBS buffer (NC).

[0123] (3) Coating: Add 1 mg / ml of collagen, positive control and negative control to the microplate, 100 μL per well, 5 replicates per group, and incubate overnight at 4°C.

[0124] (4) Blocking: Discard the supernatant, add 100 μL of 1% BSA (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.

[0125] (5) Cell seeding: Add 10g of the solution to each well. 5 3T3 / NIH cells in good condition, resuspended in D-PBS, were incubated at 37°C for 120 min. Each well was washed three times with D-PBS solution.

[0126] (6) Detection: The absorbance at OD450nm was measured using the CCK8 assay kit (manufacturer: Beyotime, product catalog number C0038). The cell adhesion degree was calculated according to the following formula. The cell adhesion rate reflects the cell adhesion ability of collagen. The higher the cell adhesion ability, the better the external environment can be provided to the cells in a short time, helping the cells to adhere.

[0127] Relative cell adhesion = {(OD1-OD0) / (OD2-OD0)}×100%.

[0128] Where: OD1: the average UV absorbance of each duplicate well of the collagen sample at 450nm; OD2: the average UV absorbance of each duplicate well of the control collagen sample at 450nm; OD0: the average UV absorbance of each duplicate well of the blank control group at 450nm.

[0129] (7) Statistical Analysis: A two-tailed t-test was used to analyze the statistical differences between the target recombinant humanized collagen and the negative control. The results are as follows: Figure 5 As shown, compared with the D-PBS group (NC), the positive control (PC) had a significant effect on promoting cell adhesion. Recombinant humanized collagen XIX-1 and recombinant humanized collagen XIX-2 also promoted cell adhesion, with cell adhesion activities of 115.3% and 127.8% respectively, which were significantly higher than those of the positive control.

[0130] Example 4: Cytotoxicity assay of recombinant XIX type humanized collagen

[0131] 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 generated is directly proportional to the number of viable cells. Therefore, this property can be used for direct HFB cytotoxicity analysis.

[0132] (1) Experimental groups: DMEM medium containing 0.4% serum and double antibiotics was used as negative control (NC), 8% NaCl was used as positive control (PC), and recombinant humanized collagen XIX-1 and XIX-2 with concentrations of 0.5, 0.75 and 1 mg / ml were used as experimental groups. Complete culture medium without cells was used as zeroing well.

[0133] (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.

[0134] (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 PBS. Cells were cultured for 24 h to allow adherence.

[0135] (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.

[0136] (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.

[0137] (6) Experimental detection: Discard the culture medium, add 100 µL (including 5 µL CCK8 solution) of double-antibody DMEM basal medium, incubate in an incubator for 4 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.

[0138] Cell viability = {(As-Ab) / (Ac-Ab)} × 100%.

[0139] In the formula:

[0140] As: Absorbance of experimental wells (including cells, culture medium, CCK8 solution and drug solution);

[0141] Ac: Absorbance of control wells (containing cells, culture medium, and CCK8 solution, but excluding drugs);

[0142] Ab: Absorbance of the blank group (including culture medium and CCK8 solution, but excluding cells and drugs).

[0143] See results Figure 6 The results showed that, compared with the negative control group (NC), the cell survival rate of the positive control group (PC) was significantly reduced, and the difference was statistically significant (two-tailed t-test statistical analysis). Experiments demonstrated that the two recombinant humanized collagen proteins XIX-1 and XIX-2 prepared in Example 1 of this invention have no cytotoxicity.

[0144] 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 consists of n repeating units; The amino acid sequence of the repeating unit is shown in SEQ ID NO.1; where n is 5.

2. The polypeptide according to claim 1, characterized in that, The repeating units are directly connected; the amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

3. Recombinant XIX type humanized collagen, characterized in that, The recombinant XIX type humanized collagen comprises the polypeptide described in any one of claims 1 to 2.

4. The recombinant XIX-type humanized collagen according to claim 3, characterized in that, The recombinant XIX type humanized collagen has cell adhesion activity.

5. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide according to any one of claims 1 to 2 or the recombinant XIX type humanized collagen according to claim 3 or 4.

6. The polynucleotide according to claim 5, characterized in that, The polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.5 or a degenerate sequence thereof.

7. The polynucleotide according to claim 5, characterized in that, The polynucleotide also includes a sequence encoding a purification tag and / or a leader sequence.

8. The polynucleotide according to claim 7, characterized in that, The purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

9. A biomaterial, characterized in that, The biomaterial comprises the polynucleotide as described in any one of claims 5-8; the biomaterial is an expression cassette, vector, or host cell.

10. The biomaterial according to claim 9, characterized in that, The carrier is an expression carrier.

11. The biomaterial according to claim 10, characterized in that, The vector also includes a control element operatively linked to the polynucleotide.

12. The biomaterial according to claim 11, characterized in that, The control element is a starter, a terminator, and / or an enhancer.

13. The biomaterial according to claim 9, characterized in that, The host cell is a bacterium, fungus, or animal cell.

14. The biomaterial according to claim 13, characterized in that, The bacteria include Escherichia coli; the fungi include yeast.

15. The biomaterial according to claim 14, characterized in that, The fungus is either Saccharomyces cerevisiae or Pichia pastoris.

16. A method for producing the polypeptide according to any one of claims 1-2 or the recombinant XIX type humanized collagen according to claim 3 or 4, characterized in that, The method includes: culturing host cells containing the polynucleotides of any one of claims 5-8, harvesting host cells and / or culture medium containing the polypeptide or the recombinant XIX type humanized collagen, and isolating the polypeptide or the recombinant XIX type humanized collagen therefrom.

17. A composition for promoting cell adhesion, characterized in that, The composition comprises one or more selected from the polypeptides of any one of claims 1 to 2, and the recombinant XIX type humanized collagen of claim 3 or 4.

18. The composition according to claim 17, characterized in that, The composition is for external use only.

19. The composition according to claim 17, characterized in that, The composition is a composition in the form of a solution, lyophilized powder, gel, sponge, or fiber.

20. The use of the polypeptide of any one of claims 1 to 2, the recombinant XIX type humanized collagen of claim 3 or 4, the polynucleotide of any one of claims 5 to 8, the biomaterial of any one of claims 9 to 15, or the composition of any one of claims 17 to 19 in the preparation of products for use in any one or more of the following: cell adhesion, tissue repair, tissue filling, or volume enhancement.

21. The use of the polypeptide of any one of claims 1 to 2, the recombinant XIX type humanized collagen of claim 3 or 4, the polynucleotide of any one of claims 5 to 8, the biomaterial of any one of claims 9 to 15, or the composition of any one of claims 17 to 19 in the preparation of cosmetic surgery products.

22. The use of the polypeptide according to any one of claims 1 to 2 or the recombinant XIX type humanized collagen according to claims 3 or 4 in promoting cell adhesion in vitro.

23. A method for promoting cell adhesion, characterized in that, The method includes the step of contacting the polypeptide of any one of claims 1-2, the recombinant XIX type humanized collagen of claim 3 or 4, or the composition of any one of claims 17-19 with cells; the promotion of cell adhesion is carried out in vitro.

24. The method according to claim 23, characterized in that, The cells in question are animal cells.

25. The method according to claim 23, characterized in that, The cells in question are mammalian cells.

26. The method according to claim 23, characterized in that, The cells are human cells.

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