Recombinant humanized type III collagen, its preparation method and application

Abstract

This invention specifically discloses recombinant humanized type III collagen, its preparation method, and its applications, relating to the field of collagen sequence optimization technology. This invention provides a type III collagen having the amino acid sequence shown in SEQ ID No. 2. The type III collagen provided by this invention not only has no significant cytotoxicity to cells but also exhibits extremely good cell proliferation-promoting effects, improves cell adhesion, and significantly promotes the proliferation of human immortalized epidermal cell lines, thus significantly promoting wound healing.

Description

Technical Field

[0001] This invention relates to the field of collagen sequence optimization technology, and in particular to recombinant humanized type III collagen, its preparation method, and its applications. Background Art

[0002] Collagen is a biological macromolecule widely found in animal connective tissues, accounting for approximately 25%–30% of the total protein in the body. It is the most widely distributed and highest-volume functional protein in mammals. Due to its excellent biocompatibility, biodegradability, low immunogenicity, and bioactivity (e.g., promoting cell proliferation, differentiation, and migration, and its use in repair and hemostasis), it is widely used in cosmetics, food, pharmaceuticals, and tissue engineering. Currently, the mainstream methods for collagen preparation fall into two main categories: animal-derived extraction and genetic engineering. Animal-derived collagen is mainly produced industrially through acid and enzymatic methods, which are relatively cheaper, but face challenges such as infection from animal-derived diseases, potential immune rejection or allergic reactions from heterologous collagen, and production capacity limitations. Genetic engineering technology produces recombinant collagen with a single component, high safety, and a controllable production process. Given the broad application prospects of collagen, especially the large demand for high-quality collagen in high-end medical materials and health products, the recombinant expression of collagen using genetic engineering technology has become a highly significant research direction.

[0003] Genetic engineering involves different expression systems such as E. coli, yeast, insect cells, mammalian cells, and transgenic crops. Expression systems such as mammalian cells and insect cells are costly and time-consuming, making it difficult to meet the needs of industrialization. In contrast, the production of recombinant collagen by microbial fermentation is low-cost, short-cycle, and easier to cultivate, making it more suitable for commercial production.

[0004] Bacterial expression systems commonly suffer from drawbacks such as: the generation of pyrogens that make the expressed products difficult to apply clinically; the expression of the target protein in the form of inclusion bodies, making product purification difficult; and the imperfect post-translational processing and modification system of prokaryotic expression systems, resulting in low biological activity of the expressed products. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] Therefore, one of the main objectives of this invention is to provide a type III collagen protein having the amino acid sequence shown in SEQ ID No. 2. The type III collagen protein provided by this invention has no significant cytotoxicity to cells, exhibits extremely good proliferative activity, significantly better cell adhesion than commercially available collagen proteins, and significantly promotes the proliferation of human immortalized epidermal cell lines, thus significantly promoting wound healing.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides a type III collagen, wherein, compared with the amino acid sequence of wild-type type III collagen, one or more of the amino acids at positions 5, 14, 20, 56, 62, 74, 83, 113, 115, 119, 149, 203, 209, 212, 233, 242, 269, 308, 311, 317, and 323 are replaced by proline; and / or

[0009] Insert glycine-glutamic acid-lysine after position 78; and / or

[0010] Insert arginine-glycine-glutamic acid after position 287; and / or

[0011] Amino acid at position 318 is missing;

[0012] The amino acid sequence of the wild-type type III collagen is shown in SEQ ID No. 5.

[0013] In one embodiment, compared with the amino acid sequence of wild-type type III collagen, amino acids at positions 5, 14, 20, 56, 62, 74, 83, 113, 115, 119, 149, 203, 209, 212, 233, 242, 269, 308, 311, 317, and 323 of the amino acid sequence of the type III collagen are replaced by proline; and

[0014] A glycine-glutamic acid-lysine sequence is inserted after position 78; and

[0015] Arginine-glycine-glutamic acid is inserted after position 287; and

[0016] The 318th amino acid is missing.

[0017] In one embodiment, the type III collagen is selected from:

[0018] (1): Type III collagen having the amino acid sequence shown in SEQ ID No. 2; or

[0019] (2): Type III collagen homologous to the amino acid sequence shown in SEQ ID NO.2 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity; or

[0020] (3): Type III collagen derived from (1) or (2) by substitution, deletion or addition of one or more amino acids in the amino acid sequence of (1) or (2).

[0021] In one embodiment, the type III collagen includes an integrin recognition site.

[0022] In one embodiment, the integrin recognition site includes GER and / or GEK.

[0023] In one embodiment, the integrin recognition site includes GER and GEK.

[0024] In one embodiment, the number of GERs is four.

[0025] In one embodiment, the number of GEKs is three.

[0026] In one embodiment, the type III collagen comprises 4 GERs and 3 GEKs.

[0027] In another aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned type III collagen.

[0028] In one embodiment, the nucleic acid molecule is selected from:

[0029] (1): A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.1; or

[0030] (2): A nucleic acid molecule that hybridizes with the sequence defined in (1) under strict conditions and has a function.

[0031] In another aspect, the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0032] In one embodiment, the vector comprises one or a combination of bacterial plasmids, bacteriophages, yeast plasmids, or animal cell viruses.

[0033] In one embodiment, the vector is a yeast plasmid.

[0034] In one embodiment, the vector is a Pichia pastoris plasmid.

[0035] In one embodiment, the carrier is pPIC9K.

[0036] In another aspect, the present invention also provides an engineered cell that expresses the aforementioned type III collagen or nucleic acid molecules or carriers.

[0037] In one embodiment, the cell is a Pichia pastoris cell.

[0038] In another aspect, the present invention also provides the application of the above-mentioned type III collagen or its promoter in promoting cell proliferation.

[0039] In one embodiment, the promoter includes a type III collagen expression vector, exogenous type III collagen, a naked nucleic acid molecule encoding a type III collagen sequence, a liposome-encapsulated nucleic acid molecule encoding a type III collagen sequence, or a type III collagen precursor protein, conjugate, or complex capable of being converted into type III collagen in vivo.

[0040] In one embodiment, the cells include fibroblasts and / or epidermal cells.

[0041] (III) Beneficial Effects

[0042] This invention provides a type III collagen. Compared with the prior art, it has the following advantages:

[0043] 1. The recombinant human type III collagen provided by this invention has a suitable molecular weight and protein modifications, such as glycosylation and hydroxylation. Simultaneously, the gene sequence contains multiple GER (characteristic sequence Gly-Glu-Arg) and GEK (characteristic sequence Gly-Glu-Lys) integrin recognition sites. These GER and GEK sites can be recognized by integrin recognition sites on the cell membrane, and subsequently transmitted to the cell via signal transduction, promoting cell proliferation and adhesion. It exhibits excellent activity, demonstrating superior cell proliferation-promoting effects not only in mouse embryonic fibroblasts but also in human immortalized epidermal cells. It can be widely applied in wound repair materials, medical aesthetics, cosmetics, health products, and other fields, with particularly promising applications in skin injuries.

[0044] 2. Based on the expression characteristics of Pichia pastoris, the nucleotide sequence of type III collagen was codon optimized and the expression vector was screened and optimized. At the same time, based on the specific triple helix structure of collagen, the amino acid sequence of recombinant human type III collagen was modified and optimized by combining artificial intelligence protein-aided design tools. This not only made the nucleotide sequence encoding recombinant human type III collagen more suitable for the Pichia pastoris expression system, but also promoted the formation of the triple helix structure of recombinant human type III collagen.

[0045] 3. The engineered bacteria constructed from the recombinant human type III collagen gene sequence designed in this invention are not easily degraded during fermentation, and the fermentation yield reaches a relatively high level of 16.30 g / L, making them more suitable for commercial or large-scale preparation. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 It is the COL3A1-KN3 amino acid sequence.

[0048] Figure 2 This is an agarose gel electrophoresis image of the COL3A1-KN3 DNA fragment.

[0049] Figure 3 This is a gel electrophoresis image of COL3A1-KN3 protein expression during the shake-flask stage.

[0050] Figure 4 This is the fermenter expression test of COL3A1-KN3 engineered bacteria, and the SDS-PAGE detection results after 108 hours of induced expression.

[0051] Figure 5 This is a fermenter expression test of the COL3A1-KN3 engineered bacteria, and a statistical analysis of the expression yield data after 108 hours of induction.

[0052] Figure 6 The results are from SDS-PAGE gel electrophoresis analysis of the COL3A1-KN3 purification process.

[0053] Figure 7 The results are from SEC-HPLC analysis of the purified COL3A1-KN3 polymer.

[0054] Figure 8 The results are obtained by circular dichroism spectroscopy after lyophilization of COL3A1-KN3.

[0055] Figure 9 This is the standard curve for detecting hydroxyproline in purified COL3A1-KN3 protein.

[0056] Figure 10 This is a cell proliferation experiment using COL3A1-KN3 protein; A shows the cell proliferation results after 24 hours of incubation; B shows the cell proliferation results after 48 hours of incubation.

[0057] Figure 11 This is an experiment on the cell adhesion of COL3A1-KN3 protein.

[0058] Figure 12 These are cell scratch experiments with different mass concentrations of COL3A1-KN3.

[0059] Figure 13This is a comparison image of SEQ ID NO.2 and SEQ ID NO.5. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] Terms and Definitions

[0062] As used herein, the terms "type III collagen," "recombinant humanized type III collagen," and "COL3A1-KN3" are used interchangeably. The type III collagen disclosed in this invention may be the type III collagen encoded by the sequence of SEQ ID NO.1 or a homologous sequence that functions with type III collagen (e.g., a homologous sequence of a protein obtainable from databases or alignment software known in the art), a variant, or a modified form. For example, the protein may be selected from:

[0063] (1): A protein having the amino acid sequence shown in SEQ ID No. 2; or

[0064] (2): Proteins homologous to the amino acid sequence shown in SEQ ID NO.2 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity; or

[0065] (3): Proteins derived from (1) or (2) by substitution, deletion or addition of one or more amino acids in the amino acid sequence of (1) or (2).

[0066] This invention discloses variations of type III collagen including (but not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein or polypeptide. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of type III collagen; for example, the type III collagen disclosed in this invention may or may not include the starting residues while still retaining its functional activity.

[0067] Type III collagen in (3) above can be obtained by random mutagenesis through radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.

[0068] Depending on the host used in the recombinant production protocol, the type III collagen disclosed in this invention may be glycosylated or non-glycosylated. The term also includes active fragments and active derivatives of the protein.

[0069] The variants of this polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences that hybridize to protein-coding sequences under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the protein. Other polypeptides, such as fusion proteins comprising a protein or a fragment thereof, may also be used in this invention. In addition to nearly full-length polypeptides, this disclosure also includes soluble fragments of the protein. Typically, the fragment has at least about 10 consecutive amino acids of the protein sequence, typically at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0070] As used herein, the terms “nucleic acid molecule,” “type III collagen encoding gene,” “nucleic acid molecule encoding type III collagen,” and “COLIII-20” are used interchangeably and all refer to a nucleotide sequence encoding the type III collagen disclosed in this invention, which may be, for example, the nucleotide sequence shown in SEQ ID NO.1, a molecule that hybridizes to these sequences under stringent conditions, or a family gene molecule that is highly homologous to the above-mentioned molecules, and the expression function of the gene.

[0071] The nucleic acid molecules disclosed in this invention are selected from:

[0072] (1): A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.2; or

[0073] (2): A nucleic acid molecule that hybridizes with the sequence defined in (1) under strict conditions and has a function.

[0074] As used herein, the term “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the similarity between the two sequences is at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, more preferably 95%. For example, the sequence may be a complementary sequence to the sequence defined in (1).

[0075] The full-length sequences or fragments of nucleic acid molecules disclosed in this invention can generally be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0076] It should be understood that the nucleic acid molecules disclosed in this invention are preferably obtained from humans. Other nucleic acid molecules obtained from other animals that are highly homologous to human nucleic acid molecules (e.g., having more than 50%, preferably more than 55%, 60%, 65%, 70%, 75%, 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more sequence identity) are also within the scope of preferred consideration in this disclosure. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0077] As used herein, the terms "promoter" or "promoter of type III collagen or its coding sequence" are used interchangeably and refer to substances that can increase the level or activity of type III collagen or its encoding nucleic acid molecule. Promoters that can be used in this invention include, but are not limited to: type III collagen expression vectors, exogenous type III collagen, naked nucleic acid molecules encoding type III collagen sequences, liposome-encapsulated nucleic acid molecules encoding type III collagen sequences, and protein precursors, conjugates, or complexes capable of being converted into type III collagen in vivo.

[0078] As used herein, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0079] Methods well known to those skilled in the art can be used to construct expression vectors containing type III collagen coding sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The pPIC9K expression system is preferably used in this invention.

[0080] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0081] Vectors containing the aforementioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells. In this invention, *Pichia pastoris* cells are preferably used as the host cell.

[0082] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0083] As used herein, "primer" refers to an oligonucleotide primer that specifically anneals to type III collagen and, under appropriate conditions, initiates DNA synthesis from it to produce the corresponding amplification product. Each of the primers discussed anneals to a target within or adjacent to the corresponding target nucleic acid molecule such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more of the target bacterial gene nucleic acids are present in the sample, one or more amplification products are produced; therefore, the presence of one or more target bacterial gene amplification products indicates the presence of a bacterial strain in the sample. The amplification product should contain a nucleic acid sequence complementary to one or more detectable probes for type III collagen.

[0084] As used herein, "probe" refers to an oligonucleotide probe that anneals specifically to the nucleic acid sequence encoding type III collagen. Each cycle step includes an amplification step, a hybridization step, and a detection step, wherein the sample is contacted with one or more detectable probes to detect the presence or absence of Pichia pastoris strains in the sample.

[0085] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0086] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0087] Example 1: Design of a recombinant human type III collagen encoding gene:

[0088] The recombinant humanized type III collagen encoding gene (COLIII-20) was designed, and its nucleotide sequence is shown in SEQ ID NO.1, with the corresponding amino acid sequence shown in SEQ ID NO.2. Figure 1 As shown. The GER and GEK active sites in the amino acid sequence of recombinant humanized type III collagen (COL3A1-KN3) are shown below. Figure 1 .

[0089] 1. Gene screening and optimization process:

[0090] 1.1. Based on the codon preference of Pichia pastoris and the AT content of the DNA sequence, online codon optimization software was used to change the corresponding original type III collagen codon bases to the bases preferred by Pichia pastoris codons.

[0091] 1.2. Repeat the characteristic sites GER and GEK in the optimized base sequence a different number of times.

[0092] 1.3. The optimized nucleotide sequence (SEQ ID NO.1) was finally synthesized by General Biotechnology Co., Ltd.

[0093] 2. Gene cloning: Specific primers were designed, and PCR was used to amplify the DNA fragment encoding COLIII-20 single strand. The PCR system is shown in Table 1.

[0094] Table 1 PCR system

[0095]

[0096] The amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown (the target band size is 602bp, and the value on the left is the molecular weight).

[0097]

[0098] SEQ ID NO.2:GAPGPKGDAGAPGPRGPPGPAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGPQGMPGPRGGLGSPGPKGPKGEPGEKGGPGPDGVPGKDGPRGPTGPIGPPGPAGQPGDKGPGPAPGPPGIAGPRGSPGERGETGPPGPAGFPGAPGPNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGPAGFPGPRGPPGPPGSNGNPGPPGPSGSPGPDGPPGPAGPTGAPGSPGVSGPKGDAGQPGEKGSPGPQGPPGAPGPLGIAGITGARGERGLAGPPGMPGPRGSPGPQGPKGPSGKPGPGLSGPRGPPGPQGLP;

[0099] SEQ ID NO.3:

[0100] LEFT PRIMER:AGGTGAGAGAGGTGGTTTGG。

[0101] SEQ ID NO.4:

[0102] RIGHT PRIMER:TTCACCTGGGGATCCTCTTG。

[0103] SEQ ID NO.6:

[0104] LEFT PRIMER:AGAGGTGGTTTGGGTTCTCC。

[0105] SEQ ID NO.7:

[0106] RIGHT PRIMER:CCACCTTCACCTTTGTCACC。

[0107] SEQ ID NO.5: GAPGGKGDAGAPGERGPPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGE GGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLP.

[0108] For example, amino acid pairings of SEQ ID NO.2 and SEQ ID NO.5 Figure 13 As shown, the upper end is SEQ ID NO.2 and the lower end is SEQ ID NO.5.

[0109] Example 2: Construction of the COL3A1-KN3 expression vector:

[0110] An EcoRI restriction site sequence was added to the 5' end of the coding gene sequence. The EcoRI recognition site is GAATTC, a specific DNA sequence with 6 base pairs. EcoRI cuts the DNA between the G and A pairs at the GAATTC recognition site, resulting in a sticky 5' end. A stop codon (UAA, UGA, or UGA) and a Not:I restriction site sequence (CGCCGG↓CG…5') were added to the 3' end of the coding gene sequence. The ↓ signifies the Not:I restriction site, which cuts the DNA double strand between G and G. Subsequently, the sequence fragment was inserted into the pPIC9K expression vector via the EcoRI and Not:I restriction sites, yielding the recombinant expression vector pPIC9K-COL3A1-KN3.

[0111] Example 3 Construction of recombinant Pichia pastoris engineered strain:

[0112] The successfully constructed recombinant expression vector plasmid pPIC9K-COL3A1-KN3 was linearized and then transformed into Pichia pastoris GS115 competent cells:

[0113] 1. Vector linearization: Linearized vectors are obtained by enzyme digestion (restriction site and endonuclease: SnaBI(TACGTA)-NotI(GCGGCCGC)).

[0114] 2. Obtaining the insert fragment: The 15-20 bp sequence at the end of the linearized vector is used as a homologous sequence and added to the 5' end of the gene-specific forward / reverse amplification primer sequences, respectively. The insert fragment with the homologous sequence is obtained by amplification using this primer pair.

[0115] 3. Recombination reaction: The linearized vector and the insert fragment are mixed in proportion and reacted at 37°C for 30 min under Exnasell catalysis to complete the recombination reaction and achieve in vitro circularization of the two linearized DNAs.

[0116] 4. Transformation of competent cells: The recombinant product is directly transformed, and hundreds of monoclonal cells will be formed on the plate for later positive screening.

[0117] 4.1. Preparation of histidine-deficient MD plates: MD plates are minimal dextrose medium + / -histidine;

[0118] 4.1.1. Heat 800ml of sterile pure water to 60℃, add 100ml of 10*YNB, 2ml of 500*Biotin, 100ml of 10*Glucose, and 10ml of 100*His, mix well to prepare reagent A and store at 4℃.

[0119] 4.1.2. Add 15g of agar powder to reagent A as described in 4.1.1, mix well, pour 3-5ml into a plate, pre-cool for half an hour, and the MD plate is ready. It can be stored at a low temperature of 4℃ for a long time for later use.

[0120] 5. Pick single colonies from the transformed MD plate and place them in 250 ml of G418 medium containing 500 μL of YPD (0.75 mg / mL). Incubate at 30°C and 240 rpm for 18–24 h. Then, transfer them sequentially to G418 medium containing 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 4.0 mg / ml, and 6.0 mg / ml of YPD for further incubation.

[0121] 6. Select a vigorous bacterial strain for fermentation, following these steps: Inoculate the bacterial culture at a 2% inoculum into a 50ml shake flask containing 10ml of BMGY medium, and incubate at 30℃ and 240rpm for 18–24h; centrifuge, discard the supernatant, resuspend in 10mL of BMMY, add 100% methanol at a volume ratio (V / V) of 1.5%, place in a 28℃ constant temperature incubator, and incubate at 240rpm for 48h, adding 100% methanol every 24h until the final methanol concentration reaches 1.5%; centrifuge, and perform SDS-PAGE analysis on the supernatant.

[0122] The results are as follows Figure 3 As shown in the figure (the values ​​on the left are molecular weight), the highest expression level of COL3A1-KN3 during the shake-flask culture stage reached 0.35 g / L, as analyzed by SDS-PAGE.

[0123] Example 4: Fermentation of recombinant Pichia pastoris engineered strain:

[0124] 1. YPD plate preparation:

[0125] 1.1 Measure 1L of distilled water into a beaker, then add 10g of yeast extract, 20g of peptone, 20g of glucose, and 15-20g of agar in sequence; stir and heat until completely dissolved to obtain the culture medium (a magnetic stirrer or water bath heating can be used).

[0126] 1.2 Adjusting pH: Use pH test paper to check the pH of the solution. Usually, no adjustment is needed (YPD's natural pH is approximately 6.5-6.8). If adjustment is required, dilute NaOH or HCl can be used to adjust the pH to 6.5.

[0127] 1.3 High-Temperature Autoclaving: Dispense the solution into Erlenmeyer flasks and seal them. Autoclave: 121℃ for 15-20 minutes. Note: Glucose is easily decomposed at high temperatures; sterilization time should not be too long.

[0128] 1.4 Preparation of YPD plates: After sterilization, cool the above culture medium to approximately 50-60°C and pour it into sterile petri dishes (approximately 15-20 mL per dish) in a laminar flow hood. After solidification at room temperature, invert the plates to prevent condensation buildup. Storage: The plates can be sealed and stored at 4°C and used within 2-4 weeks.

[0129] 2. Seed culture:

[0130] 2.1 The competent cells from Example 3 were spread onto YPD plates using an inoculation loop and cultured in a 30°C incubator until single colonies grew on the plates;

[0131] 2.2 Pick a single colony of Pichia pastoris engineered strain from a YPD plate and inoculate it into a 50 mL centrifuge tube containing 5 mL of BMGY medium. Incubate at 30°C and 240 rpm for 18–24 h. Transfer the culture at a 2% inoculum to a 1 L shake flask containing 200 mL of BMGY seed culture medium and incubate at 30°C and 240 rpm for 14–18 h until OD600 = 5–6. Inoculate 10% of the 200 mL seed culture into a 5 L fermenter (containing 2 L of BSM basal salt medium).

[0132] 2.3 Fermentation Culture: During the glycerol culture stage, the temperature was maintained at 30℃, pH 5.0, and the rotation speed was adjusted to 400–800 rpm, maintaining DO > 20%. When the glycerol in the BSM medium was depleted, at which point DO = 70–80, the glycerol feeding stage began, with a volume ratio (V / V) of 50% glycerol added to further increase the cell density. When the wet weight of the cells was approximately 180–220 g / L, glycerol addition was stopped, and the cell starvation period began. After 1 hour of starvation, the methanol feeding stage began, with the induction temperature adjusted to 26℃, and methanol added to maintain DO at around 20%. The pH was maintained throughout the fermentation process using ammonia and phosphoric acid. The fermentation broth was collected after 24 hours of induction and every 6 hours, and the supernatant was collected by centrifugation for analysis.

[0133] SDS-PAGE analysis results are as follows: Figure 4 (The values ​​on the left are molecular weights) As shown, recombinant Pichia pastoris engineered strains with high expression of type III collagen were obtained.

[0134] The growth curves of fermented cells and the expression level of the target protein are as follows: Figure 5 As shown, the yield of COL3A1-KN3 reached 10.01 g / L after 72 h of induction; the highest yield of COL3A1-KN3 was 16.30 g / L after 108 h of induction.

[0135] Example 5: Purification of COL3A1-KN3:

[0136] The fermentation broth described in Example 4 was centrifuged at ultra-high speed (17000 rpm) using a disc centrifuge, and the supernatant was collected. The supernatant was concentrated by tangential flow ultrafiltration, and then the MMC (company: Qianchun MMC, column catalog number: ZD73435) composite cation exchange column was equilibrated with 5 column volumes of binding buffer (20 mM disodium hydrogen phosphate, pH 5.52, conductivity 3.53 ms / cm). Impurities were then eluted using a gradient of washing buffer (disodium hydrogen phosphate + 1 M sodium chloride, pH 5.53, conductivity 85.7 ms / cm). The purified solution at different elution peaks was collected and analyzed by SDS-PAGE gel electrophoresis. The appearance of the target band indicates that the target protein could be eluted under the buffer gradient conditions. The resulting product is the protein stock solution, which was lyophilized into a powder for later use.

[0137] Gel electrophoresis results are as follows Figure 6 (The values ​​on the left represent molecular weight), as shown in the SEC-HPLC detection results for the polymers. Figure 7 As shown in the figure, three peaks appear at different times, indicating that the prepared collagen forms a higher-order structure with trimers.

[0138] Example 7: Circular dichroism Detection of COL3A1-KN3:

[0139] 1. Sample preparation:

[0140] The COL3A1-KN3 lyophilized powder described in Example 5 was dissolved in selected sterile water for injection, and centrifuged (12,000 rpm, 10 min) to remove insoluble matter. The concentration was quantitatively determined (UV spectrophotometry) and diluted to the target concentration.

[0141] 2. Instrument calibration:

[0142] 2.1 Power on and preheat for 30 minutes, set the nitrogen flow rate (adjust according to the wavelength), and calibrate the baseline with blank buffer (scan 3 times and take the average value).

[0143] 2.2 Inject the sample into the cuvette (avoiding air bubbles), quickly place it in the temperature-controlled sample chamber, repeat the scan 3 times, and take the average value as the final spectrum.

[0144] The circular dichroism spectroscopy results of COL3A1-KN3 after lyophilization are as follows: Figure 8 As shown, a positive peak appears at 220 nm, indicating that the protein structure has triple helix higher-order structural features.

[0145] Example 8: Analysis of COL3A1-KN3 proline hydroxylation:

[0146] The content of hydroxyproline should be quantitatively detected using an amino acid analyzer according to the method specified in YY / T1453, and the results should conform to the labeled value.

[0147] Experimental steps:

[0148] 1. Collagen standard sample processing:

[0149] Accurately weigh approximately 16.875 mg of the COL3A1-KN3 lyophilized powder described in Example 5 (accurate to 0.001 mg) into a hydrolysis flask, add 2 mL of 6 mol / L hydrochloric acid solution, mix well, and then purge the tube with high-purity nitrogen gas for approximately 3 to 4 minutes to expel air from the tube. Immediately seal the flask while it is under purging. Place the flask in an oven at 110°C for accurate hydrolysis for 23 to 24 hours.

[0150] 2. Plotting the standard curve and testing the samples:

[0151] 2.1. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 560 nm, and zero the instrument with distilled water.

[0152] 2.2. Dilute the standard solution (brand: Solarbio Cat# BC0250) with distilled water to prepare standard solutions of 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, and 0.117 μg / mL (all diluted to 7.5 μg / mL, then serially diluted).

[0153] 2.3. Perform the operation according to Table 2: (3 parallel tests for both standards and samples)

[0154] Table 2. Collagen Test Composition

[0155]

[0156] 3. Construction of the standard curve: Perform linear regression on the absorbance of the standard reference series concentrations to obtain the linear regression equation.

[0157] like Figure 9 As shown, within the linear range of 0.117-7.5 μg / mL, the absorbance of the standard solution exhibits a good linear relationship with its concentration.

[0158] 4. COL3A1-KN3 testing:

[0159] The hydroxyproline content in the test tube was determined according to the above method and conditions, and the results are shown in Table 3.

[0160] Table 3. Results of COL3A1-KN3 Hydroxyproline Content Detection

[0161]

[0162] Example 9: Effect of COL3A1-KN3 on cell proliferation:

[0163] The MTT assay was used to detect the cell proliferation effects of different collagens. The experimental groups were COL3A1-KN3 and commercially available collagen (manufacturer: Chuangjian Medical; product name: Recombinant Type III Collagen Recol3-TTA01-02SC). PBS and DMSO were set as control groups to investigate the effects of COL3A1-KN3 and commercially available collagen on the proliferation of NIH / 3T3 cells.

[0164] After adding different types of collagen to NIH / 3T3 cells, the 24-hour monitoring results showed that the proliferation capacity of NIH / 3T3 cells in the COL3A1-KN3 group was approximately 413.9%, while that in the commercially available collagen group was 359.4%. The 48-hour monitoring results showed that the proliferation capacity of NIH / 3T3 cells in the COL3A1-KN3 group was approximately 315.2%, while that in the commercially available collagen group was 310.5%. The proliferation capacity of NIH / 3T3 cells in the control group was lower than that in the experimental group (results are shown in Figure 1). Figure 10 (As shown in A and B in the diagram).

[0165] The relative cell proliferation rate and cytotoxicity grading rules shown in Table 4 were used for evaluation. A relative proliferation rate of 0 or 1 in the experimental group was considered acceptable. A relative proliferation rate of 2 in the experimental group should be evaluated in conjunction with morphological characteristics; mild toxicity or no toxicity was considered acceptable. A relative proliferation rate of 3 to 5 in the experimental group was considered unacceptable.

[0166] Table 4. Relative cell proliferation rate and cytotoxicity grading

[0167] (*: P<0.05; **: P<0.01; the same applies below)

[0168]

[0169] Therefore, COL3A1-KN3 not only has no obvious cytotoxicity to cells, but also has an extremely good proliferative effect, meeting the requirements as a biomaterial.

[0170] Example 10: Effect of COL3A1-KN3 on cell adhesion:

[0171] Cell adhesion assays for COL3A1-KN3 were performed according to the cell adhesion assay method outlined in the collagen industry standard document (YYT1849-2022). An automated cell counting program was used to count the number of fluorescently labeled cell nuclei, and the cell count was determined before and after centrifugation. The following formula was used for calculation:

[0172] V = (Nt / Nc) × 100%

[0173] In the formula:

[0174] V-Adhesion Percentage;

[0175] Nt - Cell count after centrifugation;

[0176] Nc - Cell count before centrifugation.

[0177] The relative cell adhesion ratio is calculated according to formula (B.2):

[0178] P = (V1 / V2) B.2

[0179] In the formula:

[0180] P - Relative cell adhesion ratio;

[0181] The average adhesion percentage of each duplicate well in the V1 collagen sample;

[0182] The average adhesion percentage of each replicate well in the V2 negative control.

[0183] The results were statistically analyzed using SPSS 11.0. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered statistically significant.

[0184] The results are as follows Figure 11 As shown, the cell adhesion of the COL3A1-KN3 group was significantly better than that of commercially available product 1 (manufacturer: Chuangjian Medical; product name: Recombinant Type III Collagen Recol3-TTA01-02SC) and commercially available product 2 (manufacturer: Jinbo Biotechnology; product name: Recombinant Type III Collagen ARHC III). Therefore, COL3A1-KN3 has good biological functions and is suitable for commercialization and the development of downstream products.

[0185] Example 11 Cell Scratch Assay (Collagen Repair Function Assay):

[0186] The COL3A1-KN3 cell scratch assay was performed according to the cell migration assay method in the collagen industry standard document (YYT1849-2022).

[0187] HaCaT cells were treated with high, medium, and low doses of COL3A1-KN3 for 24 h and 48 h, respectively, and the effect on cell migration ability was studied by scratch-call assay. The NC group (NC group: cell culture medium alone) included commercially available recombinant collagen 1 and commercially available recombinant collagen 2 at 0.5 mg / mL as positive controls. The COL3A1-KN3 groups had high doses of 0.5 mg / mL, medium doses of 0.05 mg / mL, and low doses of 0.005 mg / mL.

[0188] The results are as follows Figure 12As shown, after 48 hours, compared with the NC group, the effect of 0.005-0.5 mg / mL COL3A1-KN3 on the migration of human immortalized epidermal cell lines increased with increasing concentration, and the migration rate showed statistically significant differences (P<0.05). Specifically, after 48 hours of treatment with 0.5 mg / mL COL3A1-KN3, the cell migration rate was approximately (59.06±0.74)%, which was about 2.25 times higher than the NC group (26.24%). This effect was superior to commercially available recombinant collagen 1 (approximately 1.54 times) and commercially available recombinant collagen 2 (approximately 1.46 times).

[0189] In conclusion, COL3A1-KN3, within a concentration range of 0.005-0.5 mg / mL, significantly promotes the proliferation of human immortalized epidermal cell lines and wound healing.

[0190] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0191] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type III collagen, characterized in that, The amino acid sequence of the type III collagen is shown in SEQ ID No. 2; Compared to the amino acid sequence of wild-type type III collagen, amino acids at positions 5, 14, 20, 56, 62, 74, 83, 113, 115, 119, 149, 203, 209, 212, 233, 242, 269, 308, 311, 317, and 323 of the amino acid sequence of said type III collagen are replaced by proline; and Insert glycine-glutamic acid-lysine after position 78; and Arginine-glycine-glutamic acid is inserted after position 287; and Amino acid at position 318 is missing; The amino acid sequence of the wild-type type III collagen is shown in SEQ ID No.

5.

2. The type III collagen according to claim 1, characterized in that, The type III collagen contains an integrin recognition site.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the type III collagen as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecules are selected from: Nucleic acid molecules with the nucleotide sequence shown in SEQ ID NO.

1.

5. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 3 or 4.

6. The carrier according to claim 5, characterized in that, The vector includes one or a combination of bacterial plasmids, bacteriophages, yeast plasmids, or animal cell viruses.

7. An engineered cell, characterized in that, The engineered cells express the type III collagen as described in claim 1 or 2, the nucleic acid molecule as described in claim 3 or 4, or the vector as described in claim 5 or 6.

8. The use of the type III collagen as described in claim 1 or 2 in the preparation of a medicament for promoting wound healing.

9. Use of an agent comprising type III collagen as described in claim 1 or 2 in the preparation of a medicament for promoting wound healing.

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