A recombinant collagen type iii and its encoding nucleotide and preparation method

Abstract

The application provides a kind of recombinant collagen type III and its encoding nucleotide and preparation method, it is related to biomedical material technical field, the recombinant collagen type III, amino acid sequence is as shown in SEQ ID NO.1.The recombinant collagen type III is expressed in soluble form in prokaryotic system such as escherichia coli and glutamic acid clavulans bacillus by sequence optimization and functional modification, the proportion of soluble expression protein can reach more than 80% of total protein, break the technical bottleneck that previous prokaryotic system is difficult to express soluble collagen, and the production cost of the recombinant collagen type III expressed and produced by prokaryotic system is reduced, growth speed is extremely fast, and the technology is simple, suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a recombinant type III collagen, its encoding nucleotides, and a preparation method thereof. Background Technology

[0002] Collagen, as an important biomolecule, possesses properties such as biodegradability, excellent biocompatibility, and low immunogenicity. It effectively promotes cell proliferation and adhesion, playing a crucial role in tissue repair and hemostasis. Furthermore, collagen can self-assemble into a mechanically strong collagen fiber network, making it an ideal biomaterial currently widely used in food additives, cosmetic formulations, biomedical material manufacturing, and pharmaceutical development. Scientific research has identified 28 different subtypes of collagen, which can be divided into two main categories based on functional characteristics: fibroblastic collagen and non-fibroblastic collagen. Fibroblastic collagen mainly includes types I, II, III, V, VI, and XXVI. Type I collagen is the most abundant in the human body, accounting for over 85%, and is mainly distributed in tissues such as bones, skin, tendons, and cornea. Type II collagen is concentrated in cartilage, intervertebral discs, and the vitreous humor. Type III collagen is commonly found in blood vessel walls, new skin tissue, and scar repair areas.

[0003] However, collagen extracted from animal tissues has significant drawbacks: it is essentially a complex mixture of various molecular weight components, resulting in poor water solubility and insufficient biocompatibility. More seriously, due to the inherent characteristics of animal-derived collagen, it may carry immunogenic substances or potential pathogens, posing a safety risk of triggering immune rejection or disease transmission. This is particularly true for type III collagen, which is scarce in animals and often coexists with other types of collagen, presenting significant challenges in extraction, including high technical difficulty, difficulty in achieving product purity standards, and high production costs. Furthermore, physiological differences between individual animals can lead to poor batch-to-batch stability of collagen products, severely affecting their consistency and reliability in large-scale applications. Summary of the Invention

[0004] This invention aims to solve the problems of poor water solubility and insufficient biocompatibility of collagen extracted from animal tissues.

[0005] To address the above problems, this invention provides a recombinant type III collagen, its encoding nucleotides, and a preparation method thereof.

[0006] In a first aspect, the present invention provides a recombinant type III collagen, which is a recombinant humanized type III collagen, and the amino acid sequence is shown in SEQ ID NO.1.

[0007] Secondly, the present invention provides an Escherichia coli-optimized nucleotide encoding the recombinant type III collagen as described above, the optimized nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] Thirdly, the present invention provides a Corynebacterium glutamicum optimized nucleotide encoding recombinant type III collagen as described above, the optimized nucleotide sequence of Corynebacterium glutamicum is shown in SEQ ID NO.4.

[0009] Fourthly, the present invention provides a method for preparing the recombinant type III collagen as described above, comprising the following steps: S1: The nucleotide sequence encoding recombinant type III collagen; S2: The nucleotide sequence is ligated to the plasmid vector through the multiple cloning site to obtain the recombinant plasmid; S3: Take competent cells, add recombinant plasmids and culture them to obtain positive recombinant bacteria; S4: Inoculate the positive recombinant bacteria into a liquid culture medium containing antibiotics and culture until OD... 600 When the concentration reaches 0.6 to 0.8, an inducer is added to the liquid culture medium and culture is continued to obtain recombinant type III collagen.

[0010] Alternatively, the plasmid vector may be pET30a or Pxmj19.

[0011] Optionally, competent cells are Escherichia coli, Pichia pastoris, or Corynebacterium glutamicum. The competent cells are thawed on ice and then recombinant plasmids are added.

[0012] Optionally, the antibiotic is kanamycin, and in S4, the concentration of the antibiotic in the liquid culture medium is from 50 μg / mL to 100 μg / mL.

[0013] Optionally, the inducer is isopropyl thio-β-D-galactoside, and the step of adding the inducer to the liquid culture medium and continuing the culture includes: adding the inducer to the liquid culture medium and then culturing at 15 ℃ to 18 ℃ for 15 to 25 hours, or adding the inducer to the liquid culture medium and then culturing at 35 ℃ to 38 ℃ for 3 to 5 hours.

[0014] The beneficial effects of the recombinant type III collagen, its encoding nucleotide, and preparation method of this invention are as follows: In the recombinant type III collagen, the carbonyl oxygen atoms of the collagen Gly-XY tripeptide repeat sequence backbone can form a highly ordered "first hydration layer." This hydration layer improves the stability and solubility of the protein. The recombinant type III collagen has strong hydration layer capacity and a high average hydrophilicity, with an average hydrophilicity of -1.555. The transdermal permeability of collagen peptides is clearly inversely proportional to their molecular weight. The recombinant type III collagen encodes a low molecular weight collagen peptide of 15.85 kDa, which can penetrate the skin surface, and the low molecular weight greatly enhances its bioavailability. Through sequence optimization and functional modification, recombinant type III collagen can be expressed in a soluble form in prokaryotic systems such as Escherichia coli and Corynebacterium glutamicum. The proportion of soluble expressed protein can reach more than 80% of the total protein, breaking through the technical bottleneck of the previous difficulty in expressing soluble collagen in prokaryotic systems. The production cost of recombinant type III collagen produced by prokaryotic system expression is reduced, the growth rate is extremely fast, and the technology is simple, making it suitable for large-scale industrial production. Attached Figure Description

[0015] Figure 1 The graph shows the ProtScale analysis results of the amino acid sequence (SEQ ID NO.1) of the recombinant type III collagen in Example 1. Figure 2 This is a schematic diagram of the SDS-PAGE electrophoresis results of recombinant type III collagen expressed by BL21(DE3) in Example 2; Figure 3 This is a schematic diagram of the Western blot results of recombinant type III collagen expressed by BL21(DE3) in Example 2; Figure 4 This is a schematic diagram of the protein immunoblotting results of the BL21(DE3) recombinant bacteria product cultured in the 2L fermentation system provided in Example 2; Figure 5 This is a schematic diagram of the experimental results of the recombinant type III humanized collagen promoting the activity of human dermal fibroblasts provided in Example 2; Figure 6 This is a schematic diagram of the experimental results of recombinant type III humanized collagen promoting the proliferation of human keratinocytes (HaCaT cells) provided in Example 2; Figure 7 A scratch image of HaCaT cells, a human keratinocyte colony-forming cell line provided in Example 2; Figure 8 This is a schematic diagram of the scratch assay results of recombinant type III humanized collagen induced by human keratinocytes (HaCaT cells) provided in Example 2. Detailed Implementation

[0016] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first," "second," etc., mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0018] In related technologies, the acquisition of type III collagen mainly relies on extraction from animal tissues. However, this method has limitations such as high extraction difficulty, low product purity, high production costs, and insufficient batch-to-batch stability. Furthermore, animal-derived collagen may trigger immune responses and carries the potential risk of pathogen transmission. Simultaneously, naturally extracted collagen typically has poor water solubility, limiting its performance in various applications. These factors collectively constitute the current technical challenges facing the preparation and application of type III collagen.

[0019] To address the problems existing in the aforementioned related technologies, embodiments of the present invention provide a recombinant type III collagen, its encoding nucleotides, and a preparation method.

[0020] An embodiment of the present invention provides a recombinant type III collagen, which is a recombinant humanized type III collagen, and its amino acid sequence is shown in SEQ ID NO.1.

[0021] Specifically, the specific sequence of SEQ ID NO. 1 is GPKGNDGAPG KNGERGPGPG PGPQGPPGKNGETGPQGPPG PTGPGGDKGD TGPPGPQGAP GQNGEPGGKG ERGAPGEKGE GGPPGPKGND GAPGKNGERGGPGGPGPQGP PGKNGETGPQ GPPGPTGPGG DKGDTGPPGP QGAPGQNGEP GGKGERGAPG EKGEGGPPHHHHHH.

[0022] Recombinant type III collagen is a specific short-chain recombinant humanized type III collagen with a molecular weight of 15.85 kilodaltons (kDa). It is a specific short-chain humanized type III collagen that has undergone sequence optimization and functional modification. Sequence optimization and functional modification specifically include: reducing the molecular weight by removing signal peptides and truncating non-essential regions such as non-core regions; and designing different combinations of sequences with strong hydrophilicity and high stability through software analysis to ultimately enhance the probability of soluble expression of the exogenous protein. The encoded molecular weight refers to the theoretical molecular weight calculated from the protein's amino acid sequence; this value reflects the size of the protein.

[0023] In this embodiment, the carbonyl oxygen atoms of the Gly-XY tripeptide repeating sequence backbone in recombinant type III collagen can form a highly ordered "first hydration layer." This hydration layer improves the stability and solubility of the protein. Recombinant type III collagen has strong hydration layer capacity and a high average hydrophilicity, with an average hydrophilicity of -1.555. The transdermal efficiency of collagen peptides is clearly inversely proportional to their molecular weight. Recombinant type III collagen encodes a low molecular weight collagen peptide of 15.85 kDa, which can penetrate the skin surface, and the low molecular weight greatly enhances its bioavailability. Through sequence optimization and functional modification, recombinant type III collagen can be expressed in a soluble form in prokaryotic systems such as *E. coli* and *Corynebacterium glutamicum*. The proportion of soluble expressed protein can reach more than 80% of the total protein, breaking through the technical bottleneck of the difficulty in expressing soluble collagen in prokaryotic systems. The production cost of recombinant type III collagen produced by prokaryotic system expression is reduced, the growth rate is extremely fast, and the technology is simple, making it suitable for large-scale industrial production.

[0024] Another embodiment of the present invention provides an E. coli optimized nucleotide encoding the recombinant type III collagen as described above, the optimized nucleotide sequence of E. coli is shown in SEQ ID NO.2.

[0025] Specifically, the optimized nucleotide sequence for *E. coli* refers to a nucleotide sequence that has undergone codon optimization, the purpose of which is to improve the expression efficiency of recombinant type III collagen in the prokaryotic host *E. coli*. In this embodiment, the nucleotide sequence was determined using the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-optimization-tool. The sequence SEQ ID NO.1 was entered into the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-optimization-tool; the desired expression host *E. coli* was selected; the restriction enzyme sites NdeI and XhoI2 to be avoided were selected; after optimization, the nucleotide sequence encoding this recombinant protein, SEQ ID NO.2, was obtained, which encodes a recombinant type III collagen with an amino acid sequence as shown in SEQ ID NO.1 and a molecular weight of 15.85 kDa.

[0026] The specific sequence of SEQ ID NO.2 is 5'--3'.

[0027] In this embodiment, a specific recombinant type III collagen encoding nucleotide sequence adapted to the prokaryotic E. coli expression system is provided. Through codon optimization, this sequence enables translational expression of the collagen in the prokaryotic expression system, effectively addressing issues such as low translation efficiency and low expression levels in the E. coli prokaryotic expression system. The optimized sequence is designed for a recombinant type III collagen with a calculated molecular weight of 15.85 kDa and is primarily a Gly-XY repeating sequence. Its simple structure requires no special post-translational modifications, ensuring that the resulting amino acid sequence fully meets the design requirements of the target product, thus preserving the target protein's small molecular weight, good hydrophilicity, and stable hydration layer. Given that this sequence has undergone E. coli codon optimization, it can efficiently synergize with the E. coli translation system, avoiding translation interruptions or errors caused by codon mismatches. This significantly improves the expression yield of the target protein, meeting the needs of large-scale production, while also improving product purity and batch-to-batch consistency, effectively reducing the production difficulty and cost of collagen expression in the prokaryotic system.

[0028] Another embodiment of the present invention provides a yeast-optimized nucleotide encoding the recombinant type III collagen as described above, the yeast-optimized nucleotide sequence being shown in SEQ ID NO.3.

[0029] This embodiment utilizes the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-optimization-tool to determine the nucleotide sequence. The sequence SEQ ID NO.1 was input into the online codon optimization tool (https: / / sg.idtdna.com / pages / tools / codon-optimization-tool); the desired expression host yeast was selected; and the restriction enzyme sites NdeI and XhoI2 to be avoided were selected. The optimized sequence, SEQ ID NO.3, encodes the recombinant protein, thus clarifying the specific information of the optimized sequence and providing a precise sequence basis for subsequent construction of expression vectors and engineered strains in the yeast host.

[0030] The specific sequence of SEQ ID NO.3 is 5'--3'.

[0031] This embodiment provides a nucleotide sequence highly adapted to the yeast expression system. Yeast, as an important eukaryotic expression system, possesses unique advantages in protein expression. Recombinant type III collagen can be expressed not only in prokaryotic systems but also in eukaryotic systems. The provided yeast-optimized nucleotide sequence (SEQ ID NO.3) not only ensures the correctness of the amino acid sequence and structural function of the expressed recombinant type III collagen but also significantly improves the expression yield and solubility of the target protein, reducing the risk of inclusion body formation. Therefore, this technical solution provides a key technological foundation for the efficient, stable, and large-scale preparation of high-purity, high-activity recombinant type III collagen in a yeast system, thereby meeting the needs of industrial production.

[0032] Another embodiment of the present invention provides a Corynebacterium glutamicum optimized nucleotide encoding recombinant type III collagen as described above, the optimized nucleotide sequence of Corynebacterium glutamicum is shown in SEQ ID NO.4.

[0033] Specifically, the nucleotide optimized for *Corynebacterium glutamicum* refers to a nucleotide sequence that has undergone codon optimization to improve the expression efficiency of recombinant type III collagen in the prokaryotic host *Corynebacterium glutamicum*. In this embodiment, the nucleotide sequence was determined using the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-optimization-tool. The sequence SEQ ID NO.1 was entered into the online codon optimization tool (https: / / sg.idtdna.com / pages / tools / codon-optimization-tool); the desired expression host *Corynebacterium glutamicum* was selected; the restriction enzyme sites NdeI and XhoI2 to be avoided were selected; and the optimized nucleotide sequence encoding this recombinant protein, SEQ ID NO.4, was obtained.

[0034] The specific sequence of SEQ ID NO.4 is 5'--3'.

[0035] In this embodiment, a specific recombinant type III collagen encoding nucleotide sequence adapted to the prokaryotic Corynebacterium glutamicum expression system is provided. Through codon optimization, this sequence enables translational expression of the collagen in the prokaryotic expression system, effectively addressing the problems of low translation efficiency and low expression levels in the Corynebacterium glutamicum prokaryotic expression system. The optimized sequence is designed for a recombinant type III collagen with a calculated molecular weight of 15.85 kDa and is primarily a Gly-XY repeating sequence. Its simple structure requires no special post-translational modifications, ensuring that the resulting amino acid sequence fully meets the design requirements of the target product, thus preserving the target protein's small molecular weight, good hydrophilicity, and stable hydration layer. Given that this sequence has undergone codon optimization for Corynebacterium glutamicum, it can efficiently synergize with the Corynebacterium glutamicum translation system, avoiding translation interruptions or errors caused by codon mismatches. This significantly improves the expression yield of the target protein, meeting the needs of large-scale production, while also improving product purity and batch-to-batch consistency, effectively reducing the production difficulty and cost of collagen expression in the prokaryotic system.

[0036] Another embodiment of the present invention provides a method for preparing recombinant type III collagen as described above, comprising the following steps: S1: The nucleotide sequence encoding recombinant type III collagen; S2: The nucleotide sequence is ligated to the plasmid vector through the multiple cloning site to obtain the recombinant plasmid; S3: Take competent cells, add recombinant plasmids and culture them to obtain positive recombinant bacteria; S4: Inoculate the positive recombinant bacteria into a liquid culture medium containing antibiotics and culture until OD... 600 When the concentration reaches 0.6 to 0.8, an inducer is added to the liquid culture medium and culture is continued to obtain recombinant type III collagen.

[0037] In this embodiment, recombinant type III collagen was prepared using genetic engineering technology. The yield of recombinant type III collagen was increased through expanded culture. The prepared recombinant type III collagen expression avoided the potential risks of animal diseases or viruses and immune rejection reactions associated with traditional extraction methods, exhibiting excellent medical properties. Furthermore, the preparation process effectively controls production costs and product quality, and is easy to scale up. Nucleotide sequences were ligated to plasmid vectors via multiple cloning sites, ensuring the accuracy of recombinant plasmid construction. The transformation of the recombinant plasmid into competent cells was performed using ice bath, heat shock, and resuscitation culture, combined with antibiotic screening, to efficiently and accurately select successfully transformed host cells. In subsequent culture, OD was controlled... 600Induction to a logarithmic growth phase of 0.6 to 0.8 effectively enhances the expression efficiency of the target protein. The entire process is clear and controllable, ensuring a stable yield of the target product that meets design requirements, thus providing a reliable technical approach for the large-scale production of recombinant type III collagen.

[0038] Alternatively, the plasmid vector may be pET30a or Pxmj19.

[0039] Specifically, plasmid vectors serve as carriers of target genes, introducing them into host cells for replication, transcription, and translation, thereby achieving the expression of the target protein. A suitable plasmid vector should possess autonomous replication capability, multiple cloning sites, selection marker genes, and efficient expression regulatory elements to ensure the stable presence and efficient expression of the target gene in the host cell. Specifically, pET30a is a plasmid vector used in E. coli expression systems. Its characteristics include a strong promoter, enabling efficient expression of the target gene; simultaneously, pET30a typically carries an resistance gene as a selection marker, facilitating the screening of strains containing recombinant plasmids, and often includes a fusion expression tag, which is beneficial for subsequent purification of the target protein. Pxmj19 is a plasmid vector suitable for Corynebacterium glutamicum expression systems. Corynebacterium glutamicum, as an important industrial microorganism, has wide applications in the production of amino acids and nucleotides. The Pxmj19 vector adapted to Corynebacterium glutamicum can utilize the metabolic pathways and expression mechanisms of this host cell to achieve effective expression of recombinant proteins.

[0040] In this optional embodiment, several mature prokaryotic plasmid vectors suitable for different expression hosts are provided for the preparation of recombinant type III collagen. It is clarified that pET30a or Pxmj19, as prokaryotic plasmid vectors, can achieve soluble expression of recombinant type III collagen in prokaryotic vectors such as pET30a and Pxmj19, with the proportion of soluble expressed protein reaching over 80% of the total protein. This effectively solves the problems of poor expression of soluble nucleotides, low expression efficiency, and unsatisfactory protein preparation results in prokaryotic vectors. Specifically, in the *E. coli* expression system, the pET30a vector, with its efficient promoter and easy-to-purify tag, ensures stable and high-level expression of recombinant type III collagen and facilitates subsequent purification operations. The Pxmj19 vector provides a suitable tool for the *Corynebacterium glutamicum* expression system, fully utilizing the advantages of this host to achieve efficient production of recombinant proteins. The precise selection of these vectors allows the preparation process to be flexibly adjusted according to actual needs, thereby ensuring the stable existence and effective soluble expression of the target gene, which helps to stably obtain high-purity, high-yield recombinant type III collagen.

[0041] In some specific embodiments, the plasmid vector can also be the eukaryotic vector pPIC9K, a commonly used secretory expression vector in yeast expression systems, particularly Pichia pastoris. This vector can integrate the target gene into the host chromosome, thus providing high genetic stability. pPIC9K typically contains a promoter that induces high levels of expression and guides the secretion of the target protein into the culture medium, simplifying subsequent purification steps. In yeast expression systems, the pPIC9K vector ensures the genetic stability of recombinant type III collagen and simplifies the purification process through gene integration and secretory expression mechanisms.

[0042] Optionally, competent cells are Escherichia coli or Corynebacterium glutamicum, and recombinant plasmids are added after the competent cells are thawed on ice.

[0043] Specifically, Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli DH5α, and Escherichia coli TOP10F.

[0044] Before adding recombinant plasmids, competent cells need to be thawed on ice. Thawing on ice is a gentle thawing method that effectively maintains the viability and cell membrane integrity of competent cells. The preparation of competent cells usually involves cryopreservation, such as at -80°C or in liquid nitrogen. Removing competent cells from the cryogenic environment and slowly thawing them on ice before transformation avoids heat shock caused by sudden temperature changes, thereby reducing cell damage and loss of viability.

[0045] In this optional embodiment, the selectable competent cell types are clearly defined, and the processing method of competent cells before adding recombinant plasmids is standardized. Given that different types of competent cells (such as *E. coli* or *Corynebacterium glutamicum*) can adapt to different plasmid vectors (such as pET30a or Pxmj19), diverse options are provided for the expression of recombinant type III collagen, allowing the technical solution to flexibly address different expression needs and production scenarios. Simultaneously, thawing competent cells on ice before adding recombinant plasmids effectively protects cell viability and membrane integrity, significantly improves transformation efficiency, and ensures a sufficient number of positive clones. Due to these measures, this application can guarantee the stable expression of recombinant type III collagen, overcoming problems such as low transformation efficiency, unstable protein expression levels, and difficulty in adapting to different expression systems, thereby meeting the needs for large-scale stable production of recombinant type III collagen.

[0046] Optionally, the inducer is isopropyl thio-β-D-galactoside, and the step of adding the inducer to the liquid culture medium and continuing the culture includes: adding the inducer to the liquid culture medium and then culturing at 15 ℃ to 18 ℃ for 15 to 25 hours, or adding the inducer to the liquid culture medium and then culturing at 35 ℃ to 38 ℃ for 3 to 5 hours.

[0047] In this optional embodiment, isopropyl thio-β-D-galactoside is explicitly identified as the inducer, and two optional induction culture conditions are provided. This application effectively solves problems such as poor protein expression, misfolding, and inclusion body formation caused by unclear conditions during the induction expression process. Isopropyl thio-β-D-galactoside, as a highly efficient inducer, can stably initiate gene expression of recombinant type III collagen. The first low-temperature, long-time culture condition significantly improves the correct folding rate and the proportion of soluble active protein in recombinant type III collagen by reducing the protein synthesis rate, making it particularly suitable for applications requiring high protein activity. The second, higher-temperature, short-time culture condition accelerates protein synthesis, significantly shortens the production cycle, and improves overall production efficiency, making it more suitable for large-scale production requiring rapid acquisition of the target protein. The provision of these two induction strategies allows the preparation method to be flexibly selected according to different production needs, thereby stably and efficiently obtaining high-yield, high-purity, and highly active recombinant type III collagen, effectively improving the stability and adaptability of the preparation method.

[0048] Another embodiment of the present invention provides the application of the above-mentioned recombinant type III collagen in the preparation of pharmaceuticals, cosmetics or medical devices, wherein the medical devices include artificial blood vessels, hemostatic dressings, skin wound repair materials, cartilage repair materials or medical aesthetic materials.

[0049] This embodiment clarifies the specific application direction of recombinant type III collagen with an amino acid sequence as shown in SEQ ID NO.1 and a calculated molecular weight of 15.85 kDa, thus fully exploring the advantages of this recombinant type III collagen, such as low immunogenicity, high hydrophilicity, good stability, and no potential pathogen risk. Given that this recombinant type III collagen is a specific short-chain humanized collagen obtained through hydrophobicity analysis screening, it retains the core functional tripeptide repeat sequence of collagen, improving the safety and stability of the corresponding product from the raw material end and avoiding various inherent defects of naturally extracted collagen. Through sequence optimization and functional modification, recombinant type III collagen can be expressed in a soluble form in prokaryotic systems such as *E. coli* and *Corynebacterium glutamicum*, with the proportion of soluble expressed protein reaching over 80% of the total protein. This breaks through the technical bottleneck of the previous difficulty in expressing soluble collagen in prokaryotic systems. The production cost of recombinant type III collagen produced by prokaryotic system expression is reduced, the growth rate is extremely fast, and the technology is simple, making it suitable for large-scale industrial production.

[0050] The present invention will be further described below with reference to specific embodiments.

[0051] Example 1: Recombinant type III collagen (SEQ ID NO.1) was obtained.

[0052] Comparative Example 1: A recombinant humanized type III collagen polypeptide 1 was used, with the following amino acid sequence: GARGNDGARG SDGQPGPPGP PGPKGNDGAP GKNGERGGPG GPGPQGPPGK NGETGPQGPPGPTGPGGDKG DTGPPGPQGP RGSPGERGET GPPGPAGAPG QNGEPGGKGE RGAPGEKGEG GPPGPPGKDGTSGHPGPIGP PGPRGNRGER GSEGSPGHPG QPGPPGPPGA P (SEQ ID NO. 5).

[0053] Comparative Example 2: Recombinant humanized type III collagen polypeptide 2 was used, with the following amino acid sequence: GARGNDGARG SDGQPGPPGP PGAKGEVGPA GSPGSNGAPG QRGEPGPQGH AGAQGPPGPPGINGSPGGKG EMGAAGERGA PGFRGPAGPN GIPGEKGPAG ERGAPGPAGP RGAAGEPGRD GVPGGPGMRGMPGSPGGPGS DGKPGPPGSQ GESGRPGPPG PSGPRGQPGP KGNDGAPGKN GERGGPGGPG PQGPPGKNGETGPQGPPGPT GPGGDKGDTG PPGPQGTGGP PGENGKPGEP GPKGDAGAPG AKGDAGAPGE RGPPGPEGGKGAAGPPGLQG MPGERGGLGS PGPKGDKGEP GGPGADGVPG KDGPRGPTGP IGPPGPAGQP GDKGEGGAPGPRGSPGERGE TGPPGPAGAP GQNGEPGGKG ERGAPGEKGE GGPPGEPGRD GNPGSDGLPG RDGSPGGKGDRGENGSPGAP GAPGHPGPPG PVGPAGKSGD RGESGPAGSR GAPGPQGPRG DKGETGERGA AGIKGHRGFPGNPGAPGSPG PAGQQGPPGK DGTSGHPGPI GPPGPRGNRG ERGSEGSPGH PGQPGPPGPP GAP (SEQ ID NO. 6).

[0054] Comparative Example 3: Recombinant humanized type III collagen polypeptide 3 was used, with the following amino acid sequence: GERGAPGFRG PAGPNGIPGE KGPAGERGAP GPAGPRGERG APGFRGPAGP NGIPGEKGPAGERGAPGPAG PRGERGAPGF RGPAGPNGIP GEKGPAGERG APGPAGPRGE RGAPGFRGPA GPNGIPGEKGPAGERGAPGP AGPRGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGPAGPR GERGAPGFRG PAGPNGIPGEKGPAGERGAP GPAGPRGERG GERGAPGPAG PRGERGAPGF RGPAGPNGIP GEKGPAGERG APGPAGPRGE RGAPGFRGPAGPNGIPGEKG PAGERGAPGP AGPRGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGPAGPR GERGAPGFRGPAGPNGIPGE KGPAGERGAP GPAGPR (SEQ ID NO. 7).

[0055] The protein characteristics of the recombinant type III collagen from Example 1 and Comparative Examples 1-3 were analyzed using the Expasy-ProtParam software, and the results are shown in Table 1.

[0056] Table 1 Properties of Recombinant Type III Collagen

[0057] As shown in Table 1, the recombinant type III collagen of Example 1 has a total of 178 amino acids in sequence. The molecular weight was calculated to be 16312.24 using the Expasy-ProtParam software. The aliphatic index was 5.87, the hydrophilicity index was -1.555, and the instability index was 18.00. This protein is classified as a stable protein.

[0058] The hydrophilicity and hydrophobicity of the amino acid sequence (SEQ ID NO.1) of recombinant type III collagen were calculated using ProtScale, a protein physicochemical property analysis tool developed by the Swiss Institute of Bioinformatics (Expasy). Figure 1 As shown, Figure 1 Positive values ​​represent hydrophobic regions, while negative values ​​represent hydrophilic regions. Thus, the average hydrophilicity value of recombinant type III collagen is -1.555.

[0059] Example 2: Recombinant type III collagen was prepared using prokaryotic Escherichia coli.

[0060] 1. Construct recombinant type III collagen plasmid.

[0061] The pET-30a plasmid vector was used to construct an expression plasmid for the recombinant protein of *E. coli* BL21(DE3). The construction was performed according to the following procedure: The nucleotide sequence encoding recombinant type III humanized collagen (SEQ ID NO.1) was synthesized, with NdeI and XhoI multiple cloning sites at both ends. The synthesized product was separated by agarose gel electrophoresis, and the target DNA fragment was recovered using a GelExtraction Kit D2500 (Omega Bio-Tek, USA). Because the target DNA fragment of the gene synthesis product is a mixed system, direct enzyme digestion and ligation would severely reduce the success rate of recombinant plasmid construction, or even lead to complete construction failure. Therefore, agarose gel electrophoresis was used to separate nucleic acids based on molecular weight differences, and the gel was then cut and recovered using a kit, retaining only the full-length target DNA fragment and removing all impurities. The synthesized fragment and pET-30a vector were digested with NdeI and XhoI restriction endonucleases, and the synthesized fragment and vector were purified by gel extraction. The synthesized fragment and vector were assembled using T4 ligase at 37°C for 90 min. The ligation product was transformed into DH5α competent cells and cultured overnight on plates. After 24 h, single clones were picked and cultured. Plasmids were extracted using Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA), and Sanger sequencing was performed to verify the successful construction of recombinant type III collagen plasmid.

[0062] In the construction of the pET-30a vector, Escherichia coli DH5α was used as the cloning host for plasmid construction. Transformation and cell amplification were carried out using LB medium (containing 1% peptone, 0.5% yeast extract, and 1% sodium chloride per liter). When necessary, antibiotics were added at concentrations of 30 μg / mL chloramphenicol and 50 μg / mL kanamycin.

[0063] 2. Construct recombinant type III collagen-producing Escherichia coli.

[0064] BL21(DE3) competent cells were removed from the cryogenic freezer and thawed on ice. 100 ng of plasmid (the plasmid extracted in step 1 and constructed using pET-30a as the vector) was added to each cell. The cells were gently aspirated and mixed thoroughly. The cells were then placed on ice for 30-40 min, followed by heat shock at 42°C for 90 s in a water bath, and then placed on ice for 3-5 min. 100 μl of room temperature LB broth was added, and the cells were incubated on a shaker at 37°C and 200-250 rpm for 60 min. The bacterial culture was then mixed and plated onto kanamycin-resistant plates, which were inverted and incubated overnight at 37°C. Single colonies were picked and cultured. Plasmids were extracted using the Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA), and verified by Sanger sequencing. Strains with correctly sequenced plasmids were positive recombinant bacteria, indicating successful construction of recombinant *E. coli*.

[0065] 3. Expression of recombinant type III humanized collagen in Escherichia coli.

[0066] BL21(DE3) positive recombinant bacteria were inoculated into 4-6 ml LB tubes containing 100 μg / ml kanamycin and cultured in a shaker at 37°C and 250 rpm. When OD... 600 When the concentration reaches 0.6-0.8, add 0.5-1 mM IPTG to each of the two test tubes and incubate at 16°C for 20 hours or 37°C for 4 hours. The last test tube serves as a negative control. SDS-PAGE electrophoresis and Western blotting (WB) are used to detect protein expression and solubility. The SDS-PAGE electrophoresis results are shown below. Figure 2 As shown, Figure 2 In the table, 1 represents Protein marker, 2 represents BSA (1 μg), 3 represents BSA (2 μg), 4 represents uninduced whole cells, 5 represents whole cells induced at 16℃ for 20 h, 6 represents whole cells induced at 37℃ for 4 h, 7 represents uninduced cell lysis supernatant, 8 represents cell lysis supernatant induced at 16℃ for 20 h, 9 represents cell lysis supernatant induced at 37℃ for 4 h, 10 represents uninduced cell lysis pellet, 11 represents cell lysis pellet induced at 16℃ for 20 h, and 12 represents cell lysis pellet induced at 37℃ for 4 h. The results of Western blot (WB) detection are as follows: Figure 3 As shown, Figure 3 In the image, 1 represents the cell lysis supernatant after 20 hours of induction at 16℃, 2 represents the cell lysis supernatant after 4 hours of induction at 37℃, 3 represents the cell lysis pellet after 20 hours of induction at 16℃, 4 represents the cell lysis pellet after 4 hours of induction at 37℃, and 5 represents the protein marker. Figure 2 and Figure 3It is evident that recombinant BL21(DE3) bacteria express a large amount of recombinant type III collagen after the addition of IPTG inducer. This protein was detected in the cell lysis supernatant, but almost undetectable in the precipitated inclusion bodies, indicating that the protein is mainly expressed in the intracellular soluble form in E. coli BL21(DE3), with no significant expression in the inclusion bodies.

[0067] The expression levels of four different recombinant type III humanized collagen sequences (SEQ ID NO.1 of Example 1, peptide 1-SEQ ID NO.5 of Comparative Example 1, peptide 2-SEQ ID NO.6 of Comparative Example 2, and peptide 3-SEQ ID NO.7 of Comparative Example 2) in two strains of *E. coli* were detected and quantitatively evaluated using SDS-PAGE electrophoresis combined with grayscale analysis, as shown in Table 2 below. This method is a semi-quantitative approach used to estimate the accumulation of recombinant proteins in the fermentation broth. By comparing the grayscale of the target protein band with that of the standard protein band, the approximate content of the target protein in the sample can be calculated. Combined with the initial fermentation broth volume, the expression level is finally obtained in mg / L.

[0068] Table 2. Expression levels of recombinant type III humanized collagen in two Escherichia coli strains.

[0069] As shown in Table 2, the recombinant type III collagen that underwent sequence optimization and functional modification in Example 1 can be expressed in a soluble form in the prokaryotic system of Escherichia coli BL21(DE3), and the proportion of soluble expressed protein can reach 80% of the total protein.

[0070] 4. Fermentation of recombinant type III humanized collagen.

[0071] Seed culture: Inoculate 1-2% of BL21(DE3) positive recombinant bacteria into 10ml LB medium and culture for 8-10 hours. Then, inoculate 5% of each bacteria into three 100ml LB medium bottles and incubate overnight. Observe the OD the next day. 600 It is 8-10.

[0072] A 2L fermentation system (see Table 3) was inoculated at a 10% inoculum size. Specific fermentation parameters were: 37℃, initial rotation speed 700-750 rpm, aeration rate 5L / h, and pH 7.0-7.2. DO% was controlled at 50-60% before induction and 30-40% at the start of induction. OD values ​​were maintained between 1 and 2 (see Table 4). 400-500 ml / h of peptone was added as the nitrogen source, with a small amount of glycerol supplemented during the later stages of fermentation. 4-5 hours after inoculation, 0.5-1 mM IPTG was added, and induction was performed at 16℃ for 20 hours. The colony was harvested when the OD value was approximately 34-40.

[0073] Table 3. Composition of Fermentation Medium

[0074] Table 4. Nitrogen-carbon source concentrations in fed culture media

[0075] 5. Purification of recombinant type III humanized collagen.

[0076] Centrifuge the fermentation broth from step 4. Add lysis buffer to the resulting cell precipitate for lysis, then sonicate and centrifuge again. Filter the supernatant through 0.45 µm and 0.22 µm filter membranes. Perform affinity chromatography on a TA-NiFF (NTA) nickel column (equilibration buffer: 0.5 mol / L NaCl, 20 mmol / L phosphate buffer, pH 7.0; eluent: 0.5 mol / L NaCl, 20 mmol / L phosphate buffer, 100 mmol / L imidazole, pH 7.0). Collect the eluent. Desalt the eluent by ultrafiltration with 20-30 mmol / L phosphate buffer. Electrophoresis analysis showed the eluent purity to be approximately 100%. See the results below. Figure 4 , Figure 4 In the table, 1 is Protein marker; 2 is eluent (10 µl); 3 is BSA (1 µg); and 4 is BSA (2 µg). (The text abruptly ends here.) Figure 4 As can be seen, the target protein was present in the eluted sample, indicating that the recombinant type III humanized collagen had a good column loading effect. The sample was concentrated and purified after passing through a nickel column.

[0077] 6. Cell proliferation experiment of recombinant type III humanized collagen.

[0078] 200 µL of recombinant type III humanized collagen sample was added to a 96-well plate and incubated at 37°C with 5% CO2 for 2 hours for solid-phase coating (protein adsorption fixation) to simulate the extracellular matrix (ECM) environment in vivo. Wells without coating were designated as the control group. After incubation, the liquid containing excess free, unadsorbed collagen was removed, and the wells were blocked with 1% BSA-PBS for 1 hour. The BSA blocking occupied the empty spaces without disrupting the adsorption of collagen to the bottom of the wells. After washing with PBS to remove free BSA and impurities, the wells were ready for use. Finally, only a layer of firmly adsorbed recombinant type III humanized collagen remained at the bottom of the wells, which could be stably used for subsequent cell seeding and proliferation experiments.

[0079] Human dermal fibroblasts were cultured normally under conditions of 5% CO2 and 37°C. When the cells reached 60-70% confluence, they were digested with trypsin and seeded into 96-well plates pre-coated with samples, at a confluence of approximately 50%. Cell viability was measured after 3 days of culture (CCK-8 method), with a sample concentration of 2.4 µg / mL.

[0080] The results showed that cell viability was significantly greater than 100%, reaching 123.5% ± 2.0% (***P<0.001), indicating a cell proliferation-promoting effect. See details. Figure 5 .

[0081] 200 µL each of a 2.4 µg / mL recombinant type III humanized collagen sample, three other recombinant type III humanized collagen peptides (samples 1, 2, and 3 from comparative examples 1-3), and commercially available recombinant collagen (Shenzhen Baiyin Biotechnology Co., Ltd., catalog number RC 3C10C) were added to 96-well plates and incubated at 37°C with 5% CO2 for 2 hours for solid-phase coating (protein adsorption fixation) to simulate the extracellular matrix (ECM) environment in vivo. Wells without coated samples were designated as the blank group. After incubation, the liquid containing excess free, unadsorbed collagen was removed, and the plates were blocked with 1% BSA-PBS for 1 hour. The BSA blocking occupied empty spaces without disrupting the adsorption of collagen to the bottom of the wells. After washing with PBS, the plates were ready for use. Ultimately, only a layer of firmly adsorbed recombinant type III humanized collagen remained at the bottom of the wells, which could be stably used for subsequent cell proliferation experiments.

[0082] Human dermal fibroblasts were cultured normally under conditions of 5% CO2 and 37°C. When the cells reached 60-70% confluence, they were digested with trypsin and seeded into pre-coated 96-well plates at approximately 50% confluence. Cell viability was measured after 3 days of culture (CCK-8 assay). Results are shown below. Figure 5 .

[0083] Figure 5 The recombinant type III humanized collagen sample of Example 1 showed a cell viability of 123.5% ± 2.0% (***P<0.001) at a concentration of 2.4 µg / mL, which was significantly higher than the cell viability of the other three recombinant type III humanized collagen samples in Comparative Examples 1-3 (110.7% ± 1.8% (***P<0.001), 105% ± 3.1% (**P<0.01), and 101.4% ± 2.6% (**P<0.01), and also significantly higher than the cell viability of commercially available recombinant collagen (112.3% ± 1.9% (***P<0.001)). This indicates that the recombinant type III humanized collagen of Example 1 has a stronger cell proliferation-promoting effect.

[0084] 7. Cell proliferation and scratch assays of recombinant type III humanized collagen.

[0085] Cell proliferation assays were performed using human keratinocytes (HaCaT) cultured normally at 37°C with 5% CO2. When cells reached 70-80% confluence, they were digested with trypsin and seeded into 96-well plates. After 24 hours of cell adhesion, different concentrations of the test sample were added. A blank control group was set up, and the experimental group contained purified recombinant type III humanized collagen at a concentration of 120.4 µg / mL. Cell viability was measured after 24 hours of incubation (CCK-8 assay). The results showed that cell viability was significantly greater than 100% at a concentration of 120.4 µg / mL, with a viability of 120.7% ± 4.8% (***P < 0.001). See details... Figure 6 .

[0086] In the cell scratch assay, human keratinocytes (HaCaT) were cultured normally under conditions of 5% CO2 and 37°C. When the cells reached 60-70% confluence, they were digested with trypsin and seeded into 6-well plates. After 24 hours of cell adhesion, the cells in the wells reached 100% confluence. Cell scratches were created using a sterile pipette tip perpendicular to the well plate and the line. The cells were washed three times with DPBS to remove the scratched cells. Serum-free medium and different concentrations of the test sample (prepared with serum-free medium) were added. A blank control group and a positive control group (bovine type 1 collagen) were set up, with 3 parallel wells for each experimental group.

[0087] Cell culture and observation: Cells were cultured in an incubator, and samples were taken at 0h (initial) and 24h (time t). Cell migration at specific locations was observed and photographed using a biological inverted microscope. Images were taken under a 4x objective lens. Figure 7 As shown, ensure the scratches are centered and vertical, and maintain a consistent background. Figure 7 It is evident that the low concentration of recombinant type III collagen sample (0.2 µg / mL) exhibited the strongest cell adhesion and proliferation activity, superior to the higher concentration of bovine type I collagen (0.05 mg / mL). Bovine type I collagen is the recombinant collagen with the strongest proliferative capacity currently available. Therefore, this recombinant type III collagen sample has a significant effect on promoting cell scratch healing and possesses repair efficacy.

[0088] The results of relative cell migration are as follows Figure 8 As shown, Figure 8 The relative cell migration rate of the 0.05 mg / mL bovine type 1 collagen group was 105.6% ± 70.7% (*P<0.05), significantly higher than that of the blank control group; the cell migration rate of the recombinant type III collagen sample at a concentration of 0.2 µg / mL was 106.9% ± 69.1% (**P<0.01), significantly higher than that of the blank control group and better than that of the higher concentration of bovine type 1 collagen at 0.05 mg / mL, showing a significant effect in promoting cell scratch healing and possessing repair efficacy.

[0089] The amino acid sequence of the recombinant type III collagen in this application is shown in SEQ ID NO.1. This application designed a gene encoding a recombinant type III humanized collagen with a molecular weight of 15.85 kDa, constructed a prokaryotic expression strain of *E. coli*, and performed high-efficiency soluble expression. The yield of the recombinant type III humanized collagen was increased through scale-up culture in a fermenter, and protein purity was improved using strategies such as ultrafiltration and affinity chromatography. The recombinant type III humanized collagen prepared in this application avoids the potential risks of animal diseases or viruses and immune rejection reactions associated with traditional extraction methods, exhibiting excellent medical properties. Furthermore, its fermentation preparation process allows for effective control of production costs and product quality, and is easily scalable for large-scale production.

[0090] Example 3: Recombinant type III collagen-producing bacteria were prepared using the eukaryotic system Pichia pastoris.

[0091] 1. The pPIC9K plasmid vector was used to construct expression plasmids for recombinant proteins from Pichia pastoris. The construction was carried out according to the following procedure: A nucleotide sequence encoding recombinant type III humanized collagen was synthesized, with NdeI and XhoI multiple cloning sites at both ends. The synthesized product was separated by agarose gel electrophoresis, and the target DNA fragment was recovered using a Gel Extraction Kit D2500 (Omega Bio-Tek, USA). The synthesized fragment and pPIC9K vector were digested with NdeI and XhoI restriction endonucleases, and the synthesized fragment and vector were purified by gel extraction. The synthesized fragment and vector were assembled using T4 ligase at 37°C for 90 min. The ligation product was transformed into TOP10F competent cells and cultured overnight on plates. After 24 h, single colonies were picked and cultured, and plasmids were extracted using a Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA) and verified by Sanger sequencing.

[0092] The pPIC9K vector was constructed using Escherichia coli TOP10F as the cloning host for plasmid construction. Transformation and cell amplification were performed using LB medium (containing 1% peptone, 0.5% yeast extract, and 1% sodium chloride per liter). If necessary, chloramphenicol was added at a concentration of 30 μg / mL.

[0093] 2. Construct recombinant type III collagen recombinant bacteria.

[0094] Boil 1 mL of salmon sperm DNA (2 mg / L) for 5 min, then quickly cool on ice. Centrifuge competent GS115 cells at 12000 rpm for 15 s and discard the supernatant LiCl. Add 240 μL of 500 g / L PEG-3350, 36 μL of 1 mol / L LiCl, 25 μL of salmon sperm DNA, and 50 μL of linearized recombinant plasmid DNA (5-10 μg) in sequence. Vortex vigorously for 1 min to ensure the cell pellet is thoroughly mixed with the added solution. Incubate at 30 °C for 30 min. After heat shock in a 42 °C water bath for 20 min, centrifuge at 8000 rpm for 10 min to collect the cells. Resuspend the cells in 200 μL of YPD medium and incubate with shaking at 30 °C. Add 20 μL of the resuspended cells directly to an MD plate and incubate at 30 °C for 2 to 4 days, or until the colony diameter reaches 1 mm. Single clones were selected and cultured, and plasmids were extracted using the YeastPlasmid Kit (OmegaBio-Tek, USA). Sanger sequencing was then performed for verification. Strains with correctly sequenced plasmids were positive recombinant bacteria, indicating successful construction of the Pichia pastoris recombinant strain.

[0095] Example 4: Recombinant type III collagen recombinant bacteria were prepared using the prokaryotic system Corynebacterium glutamicum.

[0096] 1. The Pxmj19 plasmid vector, using the pBL1 replicon, was used to construct an expression plasmid for the recombinant protein from Corynebacterium glutamicum. The construction was performed according to the following procedure: A nucleotide sequence encoding recombinant type III humanized collagen was synthesized, with NdeI and XhoI multiple cloning sites at both ends. The synthesized product was separated by agarose gel electrophoresis, and the target DNA fragment was recovered using a Gel Extraction Kit D2500 (Omega Bio-Tek, USA). The synthesized fragment and Pxmj19 vector were digested with NdeI and XhoI restriction endonucleases, and the synthesized fragment and vector were purified by gel extraction. The synthesized fragment and vector were assembled using T4 ligase at 37°C for 90 min. The ligation product was transformed into DH5α competent cells and cultured overnight on plates. After 24 h, single colonies were picked and cultured, and plasmids were extracted using a Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA) and verified by Sanger sequencing.

[0097] In the construction of the Pxmj19 vector, Escherichia coli DH5α was used as the cloning host for plasmid construction. Transformation and cell amplification were performed using LB medium (containing 1% peptone, 0.5% yeast extract, and 1% sodium chloride per liter). When necessary, antibiotics were added at concentrations of 30 μg / mL chloramphenicol and 50 μg / mL kanamycin.

[0098] 2. Construct recombinant type III collagen recombinant bacteria.

[0099] ATCC13032 competent *Corynebacterium glutamicum* cells were removed from an ultra-low temperature freezer and thawed on ice. 100 ng of plasmid was added, and the mixture was gently aspirated and incubated on ice for 15 min. The competent cells were then transferred to a -20°C frozen electroporation cuvette (0.1 cm gap, Bio-Rad) and electroporated using a BTX electroporator at 1.8 kV, 200 Ω, and 25 μF (5 ms). After electroporation, the cells were transferred to Eppendorf tubes containing 1 mL of LBHis liquid medium (preheated to 46°C), incubated at 46°C for 6 min, and then annealed at 30°C for 1.5 h at 200 rpm. An appropriate amount of bacterial culture (electroplation efficiency approximately 10⁵ CFU / μg DNA) was evenly spread onto LBHis agar (15 g / L) plates containing the corresponding antibiotic and incubated overnight at 30°C. Colonies formed on plates containing chloramphenicol after 36 h. Single clones were selected and cultured. Plasmids were extracted using a Gram-positive bacteria plasmid mini-extraction kit (Solarbio), and verified by Sanger sequencing. Strains with correctly sequenced plasmids were confirmed to be Gram-positive recombinant bacteria, indicating successful construction of *Corynebacterium glutamicum* recombinant bacteria.

[0100] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A recombinant type III collagen, characterized in that, The protein is a recombinant humanized type III collagen, with the amino acid sequence shown in SEQ ID NO.

1.

2. An optimized nucleotide for *E. coli* encoding the recombinant type III collagen as described in claim 1, characterized in that, The optimized nucleotide sequence of the *E. coli* is shown in SEQ ID NO.

2.

3. A Corynebacterium glutamicum optimized nucleotide encoding recombinant type III collagen as described in claim 1, characterized in that, The optimized nucleotide sequence of the *Corynebacterium glutamicum* is shown in SEQ ID NO.

4.

4. A method for preparing recombinant type III collagen as described in claim 1, characterized in that, Includes the following steps: S1: Synthesize the nucleotide sequence encoding the recombinant type III collagen; S2: The nucleotide sequence is ligated to a plasmid vector via a multiple cloning site to obtain a recombinant plasmid; S3: Take competent cells, add the recombinant plasmid and culture them to obtain positive recombinant bacteria; S4: The positive recombinant bacteria are inoculated into a liquid culture medium containing antibiotics and cultured until OD... 600 When the concentration reaches 0.6 to 0.8, an inducer is added to the liquid culture medium and culture is continued to obtain recombinant type III collagen.

5. The method for preparing recombinant type III collagen according to claim 4, characterized in that, The plasmid vector is pET30a or Pxmj19.

6. The method for preparing recombinant type III collagen according to claim 5, characterized in that, The competent cells are Escherichia coli or Corynebacterium glutamicum, and the recombinant plasmid is added after the competent cells are thawed on ice.

7. The method for preparing recombinant type III collagen according to claim 4, characterized in that, The inducer is isopropyl thio-β-D-galactoside, and the step of adding the inducer to the liquid culture medium and continuing the culture includes: adding the inducer to the liquid culture medium and then culturing at 15 ℃ to 18 ℃ for 15 to 25 hours, or adding the inducer to the liquid culture medium and then culturing at 35 ℃ to 38 ℃ for 3 to 5 hours.

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