Recombinant collagen type iii and use thereof

Abstract

The application provides a kind of recombinant collagen type III and its application, it is related to genetic engineering technical field.The application improves the yield of collagen by optimizing the nucleotide sequence of the gene of interest, optimizing the culture conditions of Pichia pastoris in multiple aspects, so that it is efficiently expressed in engineering bacteria.Recombinant engineering bacteria show strong potential in improving the yield of proteins with high economic value, and are suitable for large-scale industrial production.The recombinant collagen type III of the application has high expression, simple preparation process, is suitable for medical cosmetology, wound repair and other biological material preparation, and has broad application prospect.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a recombinant type III collagen and its applications. Background Technology

[0002] Collagen is one of the most abundant structural proteins in mammals, accounting for approximately 30% of total protein content. It is widely distributed in various tissues, from the body surface of lower vertebrates to mammalian tissues, such as skin, bones, tendons, and cartilage, but is absent in plant tissues. Collagen possesses strong tensile strength, providing mechanical stability to animals.

[0003] Vertebrates possess at least 45 different collagen genes, encoding a total of 28 types of collagen. The human collagen genome also includes several collagen-like proteins, such as acetylcholinesterase, adiponectin, lectins, C1q, and type A scavenger receptors. Based on function, domain structure, and supramolecular organization, collagen can be classified into fibroblast-forming collagen, basement membrane collagen, microfibril collagen, anchoring fibrils, hexagonal reticular collagen, and fibril-associated collagen with a discontinuous triple helix structure. The vast majority of collagens are trimers composed of three polypeptide chains; these chains can be identical (homotrimeric collagen) or different (heterotrimeric collagen). Different genes encode different collagen chains, and only collagen chains of the same type can bind together.

[0004] In order of discovery, vertebrate collagen types are labeled with Roman numerals (I-XXVIII), with each type of collagen chain named after α and an Arabic numeral. There are 28 types of collagen α chains and their tissue distribution. Type I collagen is mainly found in bones, skin, tendons, and the cornea, and is a heterotrimer composed of two α1 (I) chains and one α2 (I) chain. Type II collagen is mainly distributed in cartilage, vitreous humor, and intervertebral discs; it is a homotrimer composed of three α (II) chains. Type III collagen is dominant in blood vessels, new skin, and scar tissue, and is a homotrimer composed of three identical α (III) chains. Type IV collagen forms a network structure in the basement membrane. Other types of collagen form hexagonal lattices (types VI and X), beaded fibers (type VI), anchoring fibers (type VII), and transmembrane structures. Invertebrates possess their own unique collagen genomes, which are crucial for maintaining their structural integrity. For example, the epidermis of the nematode elegans is mainly composed of cross-linked collagen, which is encoded by more than 170 collagen genes.

[0005] Compared to typical globular proteins, the amino acid composition of collagen's primary structure is quite unique. For example, type I collagen, which is the main component of skin, has very high levels of glycine (33%) and proline (13%), and also contains three uncommon amino acids: 4-hydroxyproline (9%), 3-hydroxyproline (0.1%), and 5-hydroxylysine (0.6%). These uncommon amino acids are formed by the modification of proline and lysine in the collagen polypeptide chain by proline hydroxylase and lysine hydroxylase, respectively.

[0006] In vivo, collagen primarily exists in the form of collagen fibrils. The basic structural unit of a collagen fibril is the protocollagen molecule, composed of three intertwined α-collagen chains, each containing approximately 1000 amino acid residues, with a molecular weight of about 50-55 kDa and a length of about 300 nanometers, exhibiting a typical GlyX-Y triplet repeat sequence. The X and Y positions are typically occupied by proline and hydroxyproline. Depending on the type of collagen, specific proline and lysine residues are hydroxylated through post-translational modifications. The amount of 4-hydroxyproline is crucial for the formation of intramolecular hydrogen bonds and is a core factor in maintaining the stability of the triple helix conformation; some hydroxylysine residues can also be further modified through glycosylation. The length of the triple helix portion varies greatly among different types of collagen, with the Gly-XY repeat sequence being the main motif of the protocollagen molecule. In other collagen types, these collagen domains are much shorter, and some even contain non-triple helix breaks. For example, collagen VI and X contain triple helices of approximately 200 and 460 amino acid residues, respectively. While the triple helix is ​​a key feature of all collagens and represents the main structure of fibrous collagen, the non-collagenous regions flanking the central helical portion are also essential. Typically, C-propeptide is thought to play a role in the initiation of triple helix formation, while N-propeptide is thought to be involved in the regulation of primary fibrils.

[0007] Procollagen molecules contain multiple domains, including N-terminal propeptide, N-terminal peptide, triple helix region, C-terminal peptide, and C-terminal propeptide, as well as N and C procollagen cleavage sites. The formation of this procollagen triple helix structure requires specific conditions: the three chains must be offset by one amino acid residue (approximately 2.9 Å along the helical axis), and the third residue of each repeating sequence in each chain must be close to the common helical axis. This condition is satisfied only when the smallest amino acid, Gly, is present at this position, explaining the presence of repeating sequences like Gly-XY in collagen chains. Furthermore, this axial offset of one amino acid residue makes the three chains topologically inequivalent, which is entirely different from the case of trimeric α-helical coils where the three chains are at the same axial level; these can be distinguished by the trailing chain, intermediate chain, and leading chain.

[0008] The hydrogen bond structure between collagen chains differs from the common α-helix or β-sheet hydrogen bond structure because not all peptide bonds in repeating Gly-XY polypeptide chains can form backbone-to-backbone hydrogen bonds. Early fiber diffraction models derived the correct interchain NH···O=C linkage, where the NH group of the glycine residue acts as a donor, and the C=O group of the amino acid residue at position X in the next chain acts as an acceptor (Rich and Crick II hydrogen bond topology). This topology means that each Gly-XY triplet can only form one interchain hydrogen bond. Structurally, these interchain hydrogen bonds form a "ladder" on the triple helix. Summary of the Invention

[0009] The purpose of this invention is to provide a novel recombinant type III collagen with high expression levels.

[0010] On the one hand, this application provides a recombinant type III collagen protein, wherein the amino acid sequence of the recombinant type III collagen protein includes one or more repeating units of amino acid residues, the repeating units comprising the sequence shown in SEQ ID NO.4.

[0011] Furthermore, the number of repetitions is n, where n takes the value 10 ≤ n ≤ 100.

[0012] For example, the value of n, or the upper limit of n, or the lower limit of n, can be selected from any value among 10, 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 80, 90, and 100.

[0013] Furthermore, the number of repetitions is 66.

[0014] Furthermore, the recombinant type III collagen comprises an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 95% identity with SEQ ID NO.3.

[0015] The recombinant type III collagen includes the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.3.

[0016] Those skilled in the art will understand that reasonable sequence modifications can be made to the sequence shown in SEQ ID NO:3 without affecting its collagen affinity activity, and these modified sequences should also fall within the scope of protection of this application. Such modifications include, but are not limited to: conserved amino acid substitution, partial amino acid deletion, addition, and N-terminal or C-terminal truncation; the modified peptide should still retain the collagen binding ability and specificity equivalent to the sequence in SEQ ID NO:3. Furthermore, the collagen can be chemically modified in accordance with conventional techniques, including but not limited to: cyclization, acetylation, PAS conversion, PEGylation, fatty acid modification, coupling, binding to nanocarriers, or coupling to radionuclides, small molecule compounds, nucleotides, or proteins; modifications can occur at the N-terminus, C-terminus, main chain, side chain, or specific amino acid residues of the peptide.

[0017] It is understandable that those skilled in the art can select appropriate gene editing systems and methods to complete the construction of the aforementioned recombinant type III collagen, depending on the specific circumstances.

[0018] Optionally, the recombinant type III collagen is human type III collagen.

[0019] The amino acid residue repeating units of the recombinant type III collagen are selected from wild-type human type III collagen, and the amino acid sequence of the wild-type human type III collagen is shown in SEQ ID NO.2.

[0020] On the other hand, this application also provides a biomaterial, said biomaterial comprising any one of the following A1)-A5):

[0021] A1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding said recombinant type III collagen;

[0022] A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1);

[0023] A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2);

[0024] A4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3);

[0025] A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

[0026] Further, the nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.1.

[0027] In one alternative embodiment, the collagen contains an amino acid sequence as shown in SEQ ID NO.3, and the nucleic acid molecule encoding the collagen has a nucleotide sequence as shown in SEQ ID NO.1.

[0028] Optionally, the nucleotide sequence may also contain an resistance tag or restriction enzyme sites at both ends. More preferably, the nucleotide sequence shown in SEQ ID NO.1 contains restriction enzyme sites at both ends, with the sequences being GAATTC and GCGGCCGC in the 5'-3' direction.

[0029] Those skilled in the art will recognize that the expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of the nucleic acid molecule encoding the protein can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0030] Furthermore, the expression cassette also includes a resistance gene, which is used at least for screening positive transformants. Optionally, the resistance gene is selected from one or more of the following: hygromycin resistance gene, herbicides G418 resistance gene, kanamycin resistance gene, ampicillin resistance gene, His4 gene, and blast fungicide resistance gene.

[0031] The recombinant vector described herein refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, and those skilled in the art can choose according to the specific circumstances; no excessive restrictions are imposed here. Optionally, the vector may include a nucleic acid molecule encoding the aforementioned protein, a promoter, and transcription and translation termination signals. During the preparation of the recombinant vector, the nucleic acid molecule encoding the aforementioned protein can be located within the vector so that it can be operatively linked to an appropriate expression regulatory sequence.

[0032] In one alternative implementation, the carrier is pPIC9K.

[0033] It is understandable that those skilled in the art can select appropriate gene editing systems and methods to complete the above-mentioned modification of collagen based on the actual situation.

[0034] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain, as long as they can complete the expression of the recombinant collagen described in this application. No specific strain is limited here.

[0035] In one optional embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or introduced into a recombinant microorganism using a recombinant vector. It should be noted that, as will be apparent to those skilled in the art, the nucleic acid molecule can be selectively inserted into the genome of the starting strain or can exist on a free plasmid, as long as the expression of the nucleic acid molecule or the synthesis of recombinant type III collagen can be achieved.

[0036] Furthermore, the recombinant microorganism is selected from one or more of Streptococcus, Bacillus, Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.

[0037] Optional, Pichia pastoris.

[0038] In one optional embodiment, the Pichia pastoris is Pichia pastoris GS115.

[0039] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain, as long as they can complete the expression of the recombinant type III collagen described in this application. No specific strain is limited here.

[0040] On the other hand, this application also provides a method for preparing recombinant type III collagen, the method comprising: constructing a recombinant microorganism expressing the recombinant type III collagen, and culturing the recombinant microorganism.

[0041] Furthermore, the culture conditions are: temperature 24℃-30℃, pH value 5-6.

[0042] Optionally, the temperature is 24°C and the pH value is 6.

[0043] Optionally, the method includes the following steps:

[0044] Step 1: Construct a recombinant microorganism expressing the recombinant type III collagen; the amino acid sequence of the recombinant type III collagen is as shown in SEQ ID NO.3; the recombinant microorganism is Pichia pastoris;

[0045] Step 2: Cultivate the recombinant microorganisms in a culture medium at 24℃-30℃ and pH 5-6.

[0046] Optionally, the method includes the following steps:

[0047] Step 1: Construct a recombinant microorganism expressing the recombinant type III collagen; the amino acid sequence of the recombinant type III collagen is as shown in SEQ ID NO.3; the recombinant microorganism is Pichia pastoris;

[0048] Step 2: The recombinant microorganisms are cultured in seed culture medium at 24℃-30℃ and 200-300 rpm for 20-24 hours to obtain primary seed liquid;

[0049] Step 3: Inoculate the primary seed culture into 100-500 mL of seed culture medium at an inoculation rate of 1-5% (v / v), and incubate at 24℃-30℃ and 200-300 rpm until OD reaches 100%. 600 When the concentration reaches 5-10, a secondary seed solution is obtained;

[0050] Step 3: Adjust the pH of the fermentation medium to 5-6, set the temperature to 24℃-30℃, the aeration rate to 10-30 L / min, the tank pressure to 0.01-0.05 MPa, the dissolved oxygen to 100%, and the inoculum size to 1%-5%. Inoculate the secondary seed culture into the fermentation medium, and maintain the dissolved oxygen at above 20% by adjusting the rotation speed (300 rpm-1000 rpm) and aeration rate to carry out fermentation.

[0051] Step 4: When dissolved oxygen suddenly increases and rotation speed suddenly decreases, glycerol is added at a flow rate of 10-20 mL / h·L;

[0052] Step 5: Stop adding glycerol after fermentation for 20-40 h, and add methanol at a flow rate of 10-20 mL / h·L to maintain dissolved oxygen above 20% until the product content no longer changes. This is considered the end of fermentation. Purify the supernatant after centrifugation of the fermentation broth to obtain recombinant type III collagen.

[0053] Alternatively, the method may include the following steps:

[0054] Step 1: Construct a recombinant microorganism expressing the recombinant type III collagen; the amino acid sequence of the recombinant type III collagen is as shown in SEQ ID NO.3; the recombinant microorganism is Pichia pastoris;

[0055] Step 2: The recombinant microorganisms were cultured at 24°C and 220 rpm in seed culture medium for 24 h to obtain primary seed solution;

[0056] Step 3: Inoculate the primary seed culture into the seed culture medium at an inoculation rate of 1% (v / v) and incubate at 24°C and 220 rpm until OD reaches 100%. 600When the value reaches 10, a secondary seed solution is obtained;

[0057] Step 3: Adjust the pH of the fermentation medium to 6, set the temperature to 30℃, the aeration rate to 20 L / min, the tank pressure to 0.05 MPa, and the inoculum size to 1%-5%. Inoculate the secondary seed liquid into the fermentation medium, ensuring 100% dissolved oxygen. Maintain dissolved oxygen above 20% by adjusting the rotation speed (300 rpm-1000 rpm) and aeration rate to carry out fermentation.

[0058] Step 4: When dissolved oxygen suddenly increases and rotation speed suddenly decreases, glycerol is added at a flow rate of 15 mL / h·L;

[0059] Step 5: Stop adding glycerol after 30 h of fermentation, starve the product, and then feed with methanol at a flow rate of 10 mL / h·L to maintain dissolved oxygen at more than 20% until the product content no longer changes. This is considered the end of fermentation. Purify the supernatant after centrifugation of the fermentation broth to obtain recombinant type III collagen.

[0060] The above purification process can be carried out using general methods.

[0061] In one alternative embodiment, the purification method may include nickel column affinity chromatography.

[0062] Optionally, the seed culture medium is YPD medium.

[0063] The fermentation medium includes BSM medium and PTM1 solution.

[0064] In one optional embodiment, the BSM medium is 20 L and the PTM1 solution is 87 mL.

[0065] Those skilled in the art can select the culture medium according to the actual situation.

[0066] Currently, the integration of type III collagen is difficult due to its complex and lengthy amino acid sequence. Furthermore, yeast self-repair may occur during the integration process, making it difficult for the original collagen to be accurately integrated into the yeast. In this application, a portion of the amino acid sequence of type III collagen is replicated and repeated to arrange and integrate it in its inherent manner. This not only preserves the characteristics of collagen but also facilitates integration, resulting in an engineered bacterium with high expression levels.

[0067] On the other hand, this application also provides a composition containing the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above.

[0068] The composition of this application may also contain excipients, which may be suitable solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0069] The compositions of this application can be prepared by a common method, wherein one or more diluents or carriers may be added.

[0070] One aspect of this application relates to compositions, which are any compositions capable of achieving the effects described in this application. The compositions include, but are not limited to, the simultaneous or sequential use of the components. "Simultaneous use" includes using them together in the same formulation or separately in different formulations. "Sequential use" includes using them sequentially in different formulations, with no restriction on the order of sequential use.

[0071] The concentration of recombinant type III collagen in this application may be 1 μg / mL to 1 mg / mL.

[0072] Specifically, it can be any value or range from 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, to 1 mg / mL.

[0073] Optional, 10 μg / mL.

[0074] Those skilled in the art can choose the concentration of recombinant type III collagen to use based on the actual situation, and no specific limitation is made here.

[0075] On the other hand, this application also provides the use of the recombinant type III collagen or the recombinant type III collagen prepared by the above method in any one or more of the following:

[0076] B1) Promotes cell proliferation;

[0077] B2) Promotes cell migration;

[0078] B3) Preparation of wound repair products;

[0079] B4) Promotes gel formation;

[0080] B5) Preparation of biological scaffold-related products;

[0081] B6) Improve the storage stability and / or thermal stability of collagen products;

[0082] B7) Improves cell adhesion properties.

[0083] Optionally, this application also provides the use of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in promoting cell proliferation and / or preparing products that promote cell proliferation.

[0084] Alternatively, the cells may be fibroblasts.

[0085] The recombinant type III collagen described in this application effectively promotes cell proliferation, demonstrating its non-toxicity, good safety profile, and wound repair function, making it suitable for wide application in various fields such as medicine and cosmetics. Furthermore, it has been verified that the recombinant type III collagen exhibits superior bioactivity compared to wild-type type III collagen, with a 25.1% increase in proliferation rate.

[0086] Optionally, the concentration of the recombinant type III collagen is 0.1 μg / mL to 50 μg / mL.

[0087] Alternatively, the concentration of the recombinant type III collagen is 10 μg / mL.

[0088] Optionally, this application also provides the application of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in promoting cell migration.

[0089] Alternatively, the cells may be fibroblasts.

[0090] Optionally, the concentration of the recombinant type III collagen is 10 μg / mL.

[0091] The recombinant type III collagen described in this application can effectively promote cell migration and, compared with wild-type type III collagen, can promote cell repair more quickly after cell damage, thus achieving the effect of wound repair.

[0092] Optionally, this application also provides the application of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in the preparation of wound repair products.

[0093] Optionally, this application also provides the application of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in promoting gel formation.

[0094] Optionally, the concentration of the recombinant type III collagen is 2 mg / mL.

[0095] The recombinant type III collagen described in this application has better collagen gel-forming ability than wild type III collagen. The gel prepared by it is more elastic, denser in structure, and more stable, which makes it more suitable for the preparation of biological scaffold-related products.

[0096] Optionally, this application also provides the application of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in the preparation of biological scaffold-related products.

[0097] Optionally, this application also provides the application of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in improving the storage stability and / or thermal stability of collagen products.

[0098] Alternatively, the storage stability may include triple helix structure stability.

[0099] Alternatively, the thermal stability includes stability at 25℃-95℃. Experiments have shown that the recombinant type III collagen described in this application can maintain the stability of its triple helix structure at 37℃ or 45℃, indicating that it can maintain its good biological activity during use and transportation and storage, and has broad application prospects.

[0100] Optionally, the concentration of the recombinant type III collagen is 1 mg / mL.

[0101] Optionally, this application also provides the application of the recombinant type III collagen or the recombinant type III collagen prepared by the preparation method described above in improving cell adhesion properties and / or preparing cell adhesion-promoting products.

[0102] Optionally, the cells include human skin fibroblasts (HSF) or human umbilical vein endothelial cells (HUVEC).

[0103] Optionally, the concentration of the recombinant type III collagen is 10 μg / mL.

[0104] On the other hand, this application also provides a biological scaffold, characterized in that the biological scaffold comprises the recombinant type III collagen.

[0105] The present invention has the following beneficial effects:

[0106] This invention improves collagen production by optimizing the nucleotide sequence of the target gene and the culture conditions of Pichia pastoris, resulting in efficient expression in the Pichia pastoris strain. After 72 hours of shake-flask induction, the final induced expression level reached 17 mg / L. With optimized fermentation conditions, the yield in the fermenter reached a maximum of 21.24 g / L. This recombinant engineered strain demonstrates strong potential for increasing the yield of high-value proteins and is suitable for large-scale industrial production.

[0107] The collagen of this invention has higher thermal stability, better biological activity, higher expression, and simpler operation process, making it suitable for the preparation of biomaterials for medical aesthetics, wound repair, etc., and has broad application prospects. Attached Figure Description

[0108] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0109] Figure 1 This is the pPIC9K-Collagen map in an embodiment of the present invention;

[0110] Figure 2 These are gel electrophoresis images from an embodiment of the present invention, wherein, from left to right: 1: Marker, 2: Collagen, 3: WT;

[0111] Figure 3 This is a comparison chart of optimized fermentation conditions in embodiments of the present invention. Detailed Implementation

[0112] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0113] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0114] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0115] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0116] Recombinant microorganisms: bacterial cell lines that use genetic engineering methods to achieve efficient expression of foreign genes.

[0117] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0118] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0119] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0120] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0121] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0122] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0123] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0124] The following are examples of culture medium formulations:

[0125] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, with the remainder being water, sterilized at 121℃ for 20 min, used for the culture of Escherichia coli; kanamycin concentration is 50-100 μg / mL;

[0126] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, with the remainder being water. Sterilize at 121℃ for 20 min for the culture of Escherichia coli; kanamycin concentration is 50-100 μg / mL.

[0127] YPD liquid culture medium: 20 g / L tryptone, 10 g / L yeast extract, 2% glucose, and the remainder water, sterilized at 115℃ for 25 min, used for the culture of Pichia pastoris.

[0128] YPD solid medium: YPD liquid medium with 20 g / L agar powder and 1‰ zeocin (bleomycin) added, the remainder being water, is used for the monoclonal growth of Pichia pastoris;

[0129] BMY (1L): 1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 10% YNB, 0.2% biotin, 1‰ methanol, balance water;

[0130] BMGY (1L): 1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 10% YNB, 0.2% biotin, 1‰ methanol, 2% glycerol, balance water;

[0131] MD plates: 13.4 g / L YNB (yeast nitrogen source based, amino acid-free), 4×10 -5 % Biotin (i.e., 0.04 mg / L), 20 g / L glucose, 20 g / L agar powder, with the remainder being water; sterilize at 115℃ for 25 min;

[0132] BSM medium: 26.7 mL of 85% phosphate (H3PO4), 5 g of ammonium sulfate ((NH4)2SO4), 1.5 g of magnesium sulfate (MgSO4·7H2O), 0.1 g of calcium chloride (CaCl2·2H2O), 1.0 g of potassium chloride (KCl), 10 mL of PTM1 trace element solution, with the remainder being water; sterilize at 121℃ for 20 min.

[0133] PTM1 solution: 6.0 g copper sulfate (CuSO4·5H2O), 0.08 g potassium iodide (KI), 65 g ferrous sulfate (FeSO4·7H2O), 3.0 g magnesium sulfate (MnSO4·H2O), 0.2 g sodium molybdate (Na2MoO4·2H2O), 0.02 g boric acid (H3BO3), 0.5 g cobalt chloride (CoCl2·6H2O), 20 g zinc chloride (ZnCl2), 0.1 g nickel chloride (NiCl2·6H2O), 5 mL sulfuric acid (H2SO4), diluted to 1 L with water, filtered for sterilization or sterilized at 121℃ for 20 min.

[0134] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0135] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0136] Example 1: Construction of pPIC9K-Collagen expression plasmid

[0137] Synthesis of the collagen gene: The amino acid sequence of human type III collagen (SEQ ID NO.2) was searched on Uniprot (https: / / www.uniprot.org / ). A segment of the human type III collagen sequence (SEQ ID NO.4) was extracted and modified, and then repeated 66 times to obtain recombinant type III collagen (SEQ ID NO.3). The DNA coding sequence of the recombinant type III collagen was optimized using codon optimization software to design it according to the codon preference of Pichia pastoris, resulting in the DNA sequence encoding the recombinant type III collagen of this invention (SEQ ID NO.1). The nucleotide sequence shown in SEQ ID NO.1 contains restriction enzyme sites at both ends, with the sequences being GAATTC and GCGGCCGC (5'-3' direction) respectively. The full sequence was synthesized by Shanghai Sangon Biotech and integrated into the multiple cloning site of the pPIC9K vector.

[0138] Construction and identification of recombinant plasmids:

[0139] The DNA coding sequence of recombinant type III collagen was optimized (SEQ ID NO.1) and used... EcoR I and Sal I Enzyme digestion, separation and purification by 1% agarose gel electrophoresis, ligation overnight with T4 DNA ligase, and cloning of the synthesized gene into the yeast expression vector pPIC9K to obtain the pPIC9K-Collagen plasmid. Figure 1 As shown, the synthetic collagen sequence is inserted into EcoR I and Sal I Expression occurs between sites under the control of the AOX1 promoter, and the plasmid is fused with a cleavable α-factor at the N-terminus to obtain the recombinant plasmid. The next day, the ligation product (recombinant plasmid) is used to transform E. coli DH5α competent cells, which are then plated on LB plates containing kanamycin resistance and incubated overnight at 37°C.

[0140] Example 2: Screening for positive transformants

[0141] In this example, the CW2619M rapid plasmid miniprep kit (purchased from Kangwei Century) was used to screen the positive transformants of the recombinant plasmid constructed using the method in Example 1:

[0142] The experiment was performed according to the instructions of the CW2619M rapid plasmid miniprep kit. Specifically: a single colony was inoculated into 5 mL of LB medium containing kanamycin and incubated at 37°C with shaking for 16-20 h. 600 μL of the overnight culture was transferred to an EP tube, 100 μL of Buffer L2 was added, and the tube was gently inverted 8 times. 350 mL of Buffer N3 (RNase A) was added, and the tube was inverted 10 times. The tube was centrifuged at 13000 rpm for 3 min. 1 mL of the supernatant was transferred to a new EP tube, and 500 μL of the supernatant was transferred to an adsorption column. The column was centrifuged at 13000 rpm for 15 s, and the solution was discarded. 150 μL of Buffer PB was added, and the column was centrifuged at 13000 rpm for 15 s, and the solution was discarded. 400 μL of Buffer PW (EtOH) was added, and the column was centrifuged at 13000 rpm for 1 min, and the solution was discarded. The tube was air-dried for 10 min, and 80 μL of 70°C dd H2O was added, and the column was centrifuged at 13000 rpm for 1 min. Centrifuge at rpm for 1 min and measure Nanodrop for recombinant plasmid linearization experiments.

[0143] The linearization system of the recombinant plasmid is shown in Table 1. The reaction conditions were: 37℃ water bath for 10 min; 65℃ water bath for 20 min.

[0144] Table 1 Linearization system of recombinant plasmids

[0145]

[0146] Linearized plasmids were purified using a DNA product purification kit CW2301M (purchased from Kangwei Century).

[0147] Column equilibration: Add 500 μL of BL to CB2, centrifuge at 12000 rpm for 1 min, and discard the solution. Add 5 times the volume of PB to the linearized recombinant plasmid system and mix well. Transfer the solution to CB2, centrifuge at RT for 2 min, centrifuge at 12000 rpm for 1 min, and discard the solution. Repeat once. Centrifuge again at 12000 rpm for 2 min, discard the solution; air dry for 10 min, add 40 μL of 70℃ dd H2O, centrifuge at RT for 2 min, centrifuge at 12000 rpm for 2 min, and measure Nanodrop to obtain the linearized plasmid for electroconversion experiments.

[0148] Pichia pastoris strain GS115 electroporation:

[0149] Add 5-10 μg of linearized plasmid to 80 μL of GS115 Pichia pastoris competent cells, mix well, add to an electroporation cuvette, and incubate on ice for 5 min; electroporate at 1500 V-25 μF-200 Ω; transfer the bacterial culture to 1 mL of sorbitol solution, mix well, and incubate at 30℃ and 225 rpm for 1 h; spread 50-100 μL onto MD plates and incubate at 30℃ for 48-72 h.

[0150] Screening of positive transformants (using the yeast genomic DNA extraction kit DP307-02 purchased from Tiangen):

[0151] The experiment was conducted according to the instructions of the yeast genomic DNA extraction kit. Specifically, a single colony from a zeocin-resistant MD plate was added to 5 mL of YPD medium and incubated overnight at 220 rpm and 30°C. 1 mL of the culture was centrifuged at 12000 rpm for 1 min, and the supernatant was discarded. 600 μL of sorbitol and 5 μL of 50 U Lyticase were added and mixed. The mixture was incubated at 30°C for 30 min, then centrifuged at 4100 rpm for 10 min, and the supernatant was discarded. 200 μL of GA was added to resuspend the culture, and the mixture was mixed. 4 μL of RNase A was added, and the mixture was vortexed for 15 s and incubated at RT for 5 min. 20 μL of Proteinase K was added and mixed. 220 μL of GB was added, and the mixture was inverted and mixed. The mixture was incubated at 70°C for 10 min, then centrifuged at 3000 rpm for 3 s. 220 μL of EtOH was added, and the mixture was inverted and centrifuged at 3000 rpm for 3 s. The entire solution in the EP tube was transferred to CB3 and centrifuged at 12000 rpm. Centrifuge at 12000 rpm for 30 s and discard the solution; add 500 μL GD (EtOH), centrifuge at 12000 rpm for 30 s and discard the solution; add 600 μL PW (EtOH), centrifuge at 12000 rpm for 30 s and discard the solution, repeating this operation once; centrifuge again at 12000 rpm for 2 min and discard the solution; air dry for 10 min, add 50 μL dd H2O at 70℃, reflux for 2 min, centrifuge at 120000 rpm for 2 min. Then perform PCR and gel electrophoresis to verify the results, obtaining a positive transformant strain (Collagen) with the correct sequence.

[0152] Example 3: Shake-flask induction and screening of strains with high expression levels

[0153] Based on Examples 1 and 2, the positive transformant strain (Collagen) with the correct sequence obtained in Example 2 was used as the engineered strain for shake-flask fermentation, and the engineered strain WT expressing human type III collagen (SEQ ID NO.2) was constructed in the same way as a control.

[0154] The positive transformant strain (Collagen) obtained in Example 2 and WT were placed in 40 mL of BMGY solution and cultured overnight at 30°C and 220 rpm. The OD was adjusted. 600 With a value of 6, cells were resuspended in 40 mL of BMY medium, and 160 μL of pure methanol was added to bring the final methanol concentration in the culture medium to 1% (v / v). The culture was carried out at 25°C and 220 rpm, with 160 μL of pure methanol added every 24 h to maintain the methanol concentration at 1% (v / v). OD was then measured. 600(As shown in Table 2), until the end of 72 hours, take 1 mL of bacterial culture, centrifuge at 10000 rpm for 5 min, collect the supernatant for SDS-PAGE analysis (as shown in Table 2). Figure 2 The expression level is calculated as follows: protein expression level (mg / L) = target protein concentration in fermentation supernatant (mg / mL) × 1000 (mL / L).

[0155] Table 2. Expression levels of recombinant plasmids

[0156]

[0157] Table 2 shows that wild-type human type III collagen (WT) has a low integration efficiency in Pichia pastoris due to its long and complex amino acid sequence, with an expression level of only 5 mg / L. In contrast, the recombinant collagen (Collagen) designed by the present invention through repeating units has a significantly increased expression level of 17 mg / L due to its optimized sequence structure. Collagen was selected for subsequent production.

[0158] Example 4: Optimization of fermenter culture conditions

[0159] In this embodiment, the optimized experimental steps and groupings 1#-4# are as follows:

[0160] 1#:

[0161] 1. Preparation of seed solution:

[0162] The Collagen strain preserved in Example 3 was streaked onto a plate and incubated at 30°C for 2-3 days. A single colony with good growth was picked and inoculated into 10 mL of YPD liquid medium, and incubated at 24°C and 220 rpm for approximately 24 hours. 5 mL of the above culture was then used for further expansion culture, inoculated into 500 mL of YPD liquid medium, and incubated at 24°C and 220 rpm until the seed culture OD was measured. 600 When it reaches 10, it is used as seed liquid for the upper tank.

[0163] 2. Batch fermentation:

[0164] Add 20 L of BSM medium to the fermenter, adjust the pH to 5.0 with ammonia, sterilize at 121℃ for 30 min, and after cooling, correct the dissolved oxygen to 0%. Set the temperature to 30℃, aeration rate to 20 L / min, and pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 5.0 again. Discard approximately 500 mL of medium from the fermenter through the sampling port, leaving the initial volume of the fermenter at approximately 19.5 L. Pump the seed culture from the shake flask into the fermenter at an inoculation rate of 1%-5%, correct the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. Select the "rotation speed linkage" dissolved oxygen control mode, and maintain dissolved oxygen above 20% during fermentation by adjusting the rotation speed (300 rpm-1000 rpm) and aeration rate.

[0165] 3. Glycerin-fed fermentation:

[0166] When dissolved oxygen suddenly increases and the rotation speed suddenly decreases, the glycerol feeding fermentation stage begins. The glycerol feed is added at a flow rate of 15 mL / h·L, while the internal parameters remain unchanged.

[0167] 4. Methanol-fed fermentation:

[0168] Fermentation was stopped after 30 hours (wet weight approximately 207 g / L), glycerol supplementation was halted, and dissolved oxygen rapidly recovered, initiating the methanol-fed fermentation stage. No carbon source was added to the tank for 1 hour to ensure complete glycerol depletion. After starvation, methanol was added at a constant flow rate of 10 mL / h·L, maintaining constant tank parameters and continuously monitoring fermentation parameters to maintain dissolved oxygen levels above 20%. Fermentation was completed after 132 hours, and the tank was discharged. The fermentation product was centrifuged at 8000 rpm for 20 minutes, the supernatant was collected, and stored at -20°C.

[0169] 2#:

[0170] 1. Preparation of seed solution:

[0171] The Collagen strain preserved in Example 3 was streaked onto a plate and incubated at 30°C for 2-3 days. A single colony with good growth was picked and inoculated into 10 mL of YPD liquid medium, and incubated at 24°C and 220 rpm for approximately 24 hours. 5 mL of the above culture was then used for further expansion culture, inoculated into 500 mL of YPD liquid medium, and incubated at 24°C and 220 rpm until the seed culture OD was measured. 600 When it reaches 10, it is used as seed liquid for the upper tank.

[0172] 2. Batch fermentation:

[0173] Add 20 L of BSM medium to the fermenter, adjust the pH to 6.0 with ammonia, sterilize at 121℃ for 30 min, and after cooling, correct the dissolved oxygen to 0%. Set the temperature to 30℃, aeration rate to 20 L / min, and pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 6.0 again. Discard approximately 500 mL of medium from the fermenter through the sampling port, leaving the initial volume of the fermenter at approximately 19.5 L. Pump the seed culture from the shake flask into the fermenter at an inoculation rate of 1%-5%, correct the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. Select the "rotation speed linkage" dissolved oxygen control mode, and maintain dissolved oxygen above 20% during fermentation by adjusting the rotation speed (300 rpm-1000 rpm) and aeration rate.

[0174] 3. Glycerin-fed fermentation:

[0175] When dissolved oxygen suddenly increases and the rotation speed suddenly decreases, the glycerol feeding fermentation stage begins. The glycerol feed is added at a flow rate of 15 mL / h·L, while the internal parameters remain unchanged.

[0176] 4. Methanol-fed fermentation:

[0177] Fermentation was stopped after 30 hours (wet weight approximately 207 g / L), glycerol supplementation was halted, and dissolved oxygen rapidly recovered, initiating the methanol-fed fermentation stage. No carbon source was added to the tank for 1 hour to ensure complete glycerol depletion. After starvation, methanol was added at a constant flow rate of 10 mL / h·L, maintaining constant tank parameters and continuously monitoring fermentation parameters to maintain dissolved oxygen levels above 20%. Fermentation was completed after 132 hours, and the tank was discharged. The fermentation product was centrifuged at 8000 rpm for 20 minutes, the supernatant was collected, and stored at -20°C.

[0178] 3#:

[0179] 1. Preparation of seed solution:

[0180] The Collagen strain preserved in Example 3 was streaked onto a plate and incubated at 30°C for 2-3 days. A single colony with good growth was picked and inoculated into 10 mL of YPD liquid medium and incubated at 30°C and 220 rpm for approximately 24 hours. 5 mL of the above culture was then used for further expansion culture, inoculated into 500 mL of YPD liquid medium, and incubated at 30°C and 220 rpm until the seed culture OD was measured. 600 When it reaches 10, it is used as seed liquid for the upper tank.

[0181] 2. Batch fermentation:

[0182] Add 20 L of BSM medium to the fermenter, adjust the pH to 5.0 with ammonia, sterilize at 121℃ for 30 min, and after cooling, correct the dissolved oxygen to 0%. Set the temperature to 30℃, aeration rate to 20 L / min, and pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 5.0 again. Discard approximately 500 mL of medium from the fermenter through the sampling port, leaving the initial volume of the fermenter at approximately 19.5 L. Pump the seed culture from the shake flask into the fermenter at an inoculation rate of 1%-5%, correct the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. Select the "rotation speed linkage" dissolved oxygen control mode, and maintain dissolved oxygen above 20% during fermentation by adjusting the rotation speed (300 rpm-1000 rpm) and aeration rate.

[0183] 3. Glycerin-fed fermentation:

[0184] When dissolved oxygen suddenly increases and the rotation speed suddenly decreases, the glycerol feeding fermentation stage begins. The glycerol feed is added at a flow rate of 15 mL / h·L, while the internal parameters remain unchanged.

[0185] 4. Methanol-fed fermentation:

[0186] Fermentation was stopped after 30 hours (wet weight approximately 207 g / L), glycerol supplementation was halted, and dissolved oxygen rapidly recovered, initiating the methanol-fed fermentation stage. No carbon source was added to the tank for 1 hour to ensure complete glycerol depletion. After starvation, methanol was added at a constant flow rate of 10 mL / h·L, maintaining constant tank parameters and continuously monitoring fermentation parameters to maintain dissolved oxygen levels above 20%. Fermentation was completed after 132 hours, and the tank was discharged. The fermentation product was centrifuged at 8000 rpm for 20 minutes, the supernatant was collected, and stored at -20°C.

[0187] 4#:

[0188] 1. Preparation of seed solution:

[0189] The Collagen strain preserved in Example 3 was streaked onto a plate and incubated at 30°C for 2-3 days. A single colony with good growth was picked and inoculated into 10 mL of YPD liquid medium and incubated at 30°C and 220 rpm for approximately 24 hours. 5 mL of the above culture was then used for further expansion culture, inoculated into 500 mL of YPD liquid medium, and incubated at 30°C and 220 rpm until the seed culture OD was measured. 600 When it reaches 10, it is used as seed liquid for the upper tank.

[0190] 2. Batch fermentation:

[0191] Add 20 L of BSM medium to the fermenter, adjust the pH to 6.0 with ammonia, sterilize at 121℃ for 30 min, and after cooling, correct the dissolved oxygen to 0%. Set the temperature to 30℃, aeration rate to 20 L / min, and pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 6.0 again. Discard approximately 500 mL of medium from the fermenter through the sampling port, leaving the initial volume of the fermenter at approximately 19.5 L. Pump the seed culture from the shake flask into the fermenter at an inoculation rate of 1%-5%, correct the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. Select the "rotation speed linkage" dissolved oxygen control mode, and maintain dissolved oxygen above 20% during fermentation by adjusting the rotation speed (300 rpm-1000 rpm) and aeration rate.

[0192] 3. Glycerin-fed fermentation:

[0193] When dissolved oxygen suddenly increases and the rotation speed suddenly decreases, the glycerol feeding fermentation stage begins. The glycerol feed is added at a flow rate of 15 mL / h·L, while the internal parameters remain unchanged.

[0194] 4. Methanol-fed fermentation:

[0195] Fermentation was stopped after 30 hours (wet weight approximately 207 g / L), glycerol supplementation was halted, and dissolved oxygen rapidly recovered, initiating the methanol-fed fermentation stage. No carbon source was added to the tank for 1 hour to ensure complete glycerol depletion. After starvation, methanol was added at a constant flow rate of 10 mL / h·L, maintaining constant tank parameters and continuously monitoring fermentation parameters to maintain dissolved oxygen levels above 20%. Fermentation was completed after 132 hours, and the tank was discharged. The fermentation product was centrifuged at 8000 rpm for 20 minutes, the supernatant was collected, and stored at -20°C.

[0196] The method for calculating expression levels is the same as in Example 3, and the results are shown in Table 3 and... Figure 3 As shown.

[0197] Table 3 Expression levels under different culture conditions

[0198]

[0199] according to Figure 3 As shown, the fermentation results indicate that the protein expression level was highest when the fermentation conditions for No. 2 were a culture temperature of 24℃ and the pH of the solution was adjusted to 6.0 with ammonia.

[0200] Example 5: Collagen Functional Verification Experiment

[0201] The purpose of this experiment was to highlight the high activity and high expression characteristics of recombinant type III collagen. Therefore, the strains used in subsequent experiments were:

[0202] Group 1 (blank control): Pichia pastoris GS115 strain without any foreign genes introduced;

[0203] Group 2 (experimental group): Recombinant Pichia pastoris strain Collagen expressing the recombinant type III collagen of this application (amino acid sequence as shown in SEQ ID NO.3) (construction method is described in Examples 1-2);

[0204] Group 3 (wild-type control, WT): a recombinant Pichia pastoris strain expressing wild-type human type III collagen (amino acid sequence as shown in SEQ ID NO. 2), whose construction process was completely consistent with that of Group 2 (same vector pPIC9K, same host GS115, obtained through plasmid construction-linearization-electroporation-positive screening).

[0205] Experiment 1: The promoting effect of recombinant type III collagen on fibroblast proliferation (core function of wound repair)

[0206] 1. Experimental objective: To verify whether recombinant type III collagen can promote the proliferation of skin fibroblasts (key cells for wound repair) and to demonstrate its wound repair-related activity.

[0207] 2. Experimental materials:

[0208] 2.1 Strains and Sample Preparation:

[0209] Group 1 sample: Pichia pastoris strain GS115 without exogenous gene transfer was cultured for 72 h using the shake flask induction method in Example 3. The fermentation supernatant was collected, filtered through a 0.22 μm filter membrane for sterilization, and used as the blank control supernatant (without collagen).

[0210] Group 2 samples: Take the recombinant Pichia pastoris strain expressing recombinant type III collagen (SEQ ID NO.3) (construction method is described in Examples 1-2), culture it for 72 h as described above, collect the supernatant and purify it (using nickel column affinity chromatography, the eluent is PBS containing 200 mM imidazole, the concentration is measured after dialysis and desalting), and adjust it to 1 mg / mL for later use.

[0211] Group 3 sample: Take the recombinant Pichia pastoris strain expressing wild-type human type III collagen (SEQ ID NO.2) (construction process is the same as Group 2), culture for 72 h in the same way as above, collect the supernatant and purify it, and adjust the concentration to 1 mg / mL for later use.

[0212] 2.2 Cells: Human skin fibroblasts (HSF, ATCC number CRL-2522), cultured to the 5th generation for experiments.

[0213] 2.3 Culture media: DMEM high glucose medium (containing 4 mM L-glutamine), fetal bovine serum (FBS, Gibco), and serum-free DMEM medium.

[0214] 2.4 Reagents: MTT solution (5 mg / mL, dissolved in PBS, sterilized by 0.22 μm filtration), DMSO (analytical grade), PBS buffer (pH 7.4).

[0215] 2.5 Instruments: 96-well cell culture plates (Corning), CO2 incubator (Thermo, 37℃, 5% CO2), microplate reader (BioTek, detection wavelength 490 nm), clean bench, centrifuge.

[0216] 3. Experimental steps:

[0217] 3.1 Cell Seeding: HSF cells were resuspended in DMEM medium containing 10% FBS and the cell density was adjusted to 5 × 10⁶ cells / year. 3 100 μL of cells were seeded into each well of a 96-well plate and cultured at 37°C with 5% CO2 for 24 h. Microscopic examination confirmed that the cell adhesion rate was ≥90%.

[0218] 3.2 Sample concentration gradient setting: The purified collagen from Group 2 and Group 3 was diluted to 0.1 μg / mL, 1 μg / mL, 10 μg / mL and 50 μg / mL respectively (solvent was serum-free DMEM medium). The fermentation supernatant of Group 1 sample was directly taken after sterilization (containing 0.1% BSA to exclude nutritional differences). A basic blank group was set up (serum-free DMEM medium only).

[0219] 3.3 Grouping: Discard the original culture medium in the 96-well plate, add 100 μL of sample treatment solution to each well, and group as follows (6 replicates per group, 3 times):

[0220] Basic control group: serum-free DMEM;

[0221] Group 1: 100 μL Group 1 fermentation supernatant;

[0222] Group 3-0.1: Serum-free DMEM containing 0.1 μg / mL Group 3 collagen;

[0223] Group 3-1: Serum-free DMEM containing 1 μg / mL Group 3 collagen;

[0224] Groups 3-10: Serum-free DMEM containing 10 μg / mL Group 3 collagen;

[0225] Group 3-50: Serum-free DMEM containing 50 μg / mL Group 3 collagen;

[0226] Group 2-0.1: Serum-free DMEM containing 0.1 μg / mL Group 2 collagen;

[0227] Group 2-1: Serum-free DMEM containing 1 μg / mL Group 2 collagen;

[0228] Group 2-10: Serum-free DMEM containing 10 μg / mL Group 2 collagen;

[0229] Group 2-50: Serum-free DMEM containing 50 μg / mL Group 2 collagen.

[0230] 3.4 Culture and Detection: The 96-well plate was placed in a 37℃, 5% CO2 incubator, and detection was performed at 24 h, 48 h, and 72 h of culture: 20 μL of MTT solution was added to each well, gently shaken, and cultured for another 4 h; the liquid in the well was discarded, and 150 μL of DMSO was added to each well, and the plate was shaken horizontally for 10 min until the crystals were completely dissolved; the OD value at 490 nm was measured using a microplate reader, and the data were recorded. The formula for fibroblast proliferation rate is shown below, and the results are shown in Table 4. Significance analysis was performed on the data.

[0231] .

[0232] Table 4. Statistical results of fibroblast proliferation rate (%, mean ± standard deviation, n=6)

[0233]

[0234] 4. Results Analysis and Conclusions

[0235] 4.1 Blank control verification: The proliferation rate of Group 1 (GS115) was not significantly different from that of the basic blank group (P>0.05), indicating that the host strain itself has no proliferation-promoting activity, and the experimental results are directly caused by collagen expression.

[0236] 4.2 High Activity Comparison (Group2 vs Group3):

[0237] At a concentration of 10 μg / mL, the proliferation rate of Group 2 was significantly higher than that of Group 3 (wild type) at 24 h (152.6% vs 120.1%), 48 h (220.4% vs 180.3%), and 72 h (250.7% vs 200.5%), and the difference was extremely significant (P<0.01).

[0238] Even at a concentration of 50 μg / mL, the proliferation rate of Group 2 was still higher than that of Group 3 (235.6% vs 190.1%, P<0.05), further demonstrating the activity advantage of recombinant collagen.

[0239] 4.3 Determination of optimal concentration: 10 μg / mL was the optimal concentration for promoting proliferation in Group 2, with a proliferation rate of 250.7% after 72 h, which was 25.1% higher than that of wild type (Group 3).

[0240] 4.4 Data Reliability Verification

[0241] All groups had 6 replicates, and the experiment was repeated 3 times. Data are presented as mean ± standard deviation to ensure statistical reliability.

[0242] Differences between groups were assessed using one-way ANOVA combined with LSD-t test. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant, which is consistent with the statistical rigor of biological experiments.

[0243] Experiment 2: The promoting effect of recombinant type III collagen on fibroblast migration (a key indicator of wound healing)

[0244] 1. Experimental objective: To verify whether the recombinant type III collagen (Group2) of this application has stronger fibroblast migration-promoting activity compared with wild-type human type III collagen (Group3) and blank control (Group1), to simulate the ability of cells to aggregate to the wound during wound healing, and to prove its high activity characteristics.

[0245] 2. Experimental materials:

[0246] 2.1 Strains and Sample Preparation

[0247] Group 1 sample: Pichia pastoris strain GS115 without exogenous gene transfer was cultured for 72 h using the shake flask induction method in Example 3. The fermentation supernatant was collected, filtered through a 0.22 μm filter membrane to remove bacteria, and used as blank control supernatant (containing 0.1% BSA to exclude nutritional differences).

[0248] Group 2 sample: Recombinant Pichia pastoris strain expressing recombinant type III collagen (SEQ ID NO.3) was cultured for 72 h using the same method as described above, and the concentration was adjusted to 10 μg / mL after purification (the solvent was serum-free DMEM, the optimal proliferation concentration is the same as the results of Experiment 1 in Example 5).

[0249] Group 3 samples: Recombinant Pichia pastoris strain expressing wild-type human type III collagen (SEQ ID NO.2) was cultured for 72 h using the same method as described above, and the concentration was adjusted to 10 μg / mL after purification (solvent was serum-free DMEM).

[0250] 2.2 Cells and Reagents

[0251] Cells: Human skin fibroblasts (HSF, ATCC number CRL-2522), cultured to passage 5 for use in experiments.

[0252] Reagents: DMEM medium containing 10% FBS, serum-free DMEM medium, PBS buffer (pH 7.4), and sterile 200 μL pipette tips.

[0253] 2.3 Instruments

[0254] 24-well cell culture plate (Corning), CO2 incubator (Thermo, 37℃, 5% CO2), inverted microscope.

[0255] 3. Experimental Procedure

[0256] 3.1 Cell Plating and Synchronization

[0257] HSF cells were resuspended in DMEM medium containing 10% FBS and the cell density was adjusted to 2 × 10⁶ cells / year. 5 500 μL of cells were seeded into each well of a 24-well plate and cultured at 37°C with 5% CO2 for 24 h. Microscopic examination confirmed that the cell confluence reached 80%-90%.

[0258] 3.2 Scratch Marking and Standardization

[0259] Use a sterile 10 μL pipette tip to make three parallel scratches (spaced ≥ 5 mm) in the center of each well, with the scratch length covering 2 / 3 of the well diameter.

[0260] Rinse gently three times with PBS to remove detached cells, discard the supernatant, and ensure that no suspended cells remain in the scratched area.

[0261] 3.3 Group treatment: Add 500 μL of treatment solution to each well, and divide into groups as follows (6 replicates per group, 3 times):

[0262] Blank control group: serum-free DMEM;

[0263] Group 1: 500 μL Group 1 fermentation supernatant;

[0264] Group 2: Serum-free DMEM containing 10 μg / mL Group 2 collagen;

[0265] Group 3: Serum-free DMEM containing 10 μg / mL Group 3 collagen.

[0266] 3.4 Observation and Image Acquisition

[0267] The three scratches in each well were photographed at 100× magnification using an inverted microscope at 0 h, 12 h, 24 h, and 48 h.

[0268] 3.5 Calculation of Healing Rate

[0269] Open the image with ImageJ software, draw straight lines along both sides of the scratch, and measure the width of each scratch (measure at 0 h and 3 locations at each time point, and take the average value).

[0270] Healing rate (%) = (0 h scratch width - scratch width at a certain time point) / 0 h scratch width × 100%.

[0271] Statistical methods: One-way ANOVA was performed using GraphPad Prism 9. LSD-t test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0272] 4. The experimental results are shown in Table 5.

[0273] Table 5

[0274]

[0275] 5. Results Analysis and Conclusions

[0276] Blank control verification: There was no significant difference in healing rate between Group 1 and the blank control group (P>0.05), indicating that the host strain itself has no migration-promoting activity, and the experimental results are directly mediated by collagen.

[0277] 5.1 Comparison of migration activity (Group2 vs Group3)

[0278] At 12 h, the healing rate of Group 2 (48.2%) was significantly higher than that of Group 3 (35.6%, P<0.05), indicating that recombinant collagen can initiate cell migration earlier;

[0279] At 24 h, the healing rate of Group 2 (75.6%) was 30% higher than that of Group 3 (58.2%), and the difference was extremely significant (P<0.01).

[0280] At 48 h, the healing rate of Group 2 reached 88.5% (≥80%), which was significantly higher than that of Group 3 (72.3%), while the healing rate of the blank control group was only 45.2% (≤50%), meeting the criteria for "excellent activity performance".

[0281] 5.2 Cell morphology observation

[0282] At 48 h, a large number of spindle-shaped migrating cells were observed in the scratch area of ​​Group 2, with well-developed pseudopodia and dense arrangement; while the density of migrating cells in Group 3 was low, and only a small number of cells migrated in the blank control group.

[0283] 6. Data reliability

[0284] Each group has 6 replicates, and each scratch is measured at 3 locations. The experiment is repeated 3 times, and the data are presented as "mean ± standard deviation" to reduce random errors.

[0285] The scratches were created using standardized procedures (same batch of gun tips, fixed angle) to ensure consistent initial widths (no significant difference in scratch widths at 0 h among groups, P>0.05).

[0286] Statistical analysis showed that the differences between groups were statistically significant (P<0.01), which fully demonstrates that the migration-promoting activity of recombinant type III collagen (Group2) was significantly better than that of wild type (Group3).

[0287] Experiment 3: Collagen gel-forming ability of recombinant type III collagen (basic properties of biomaterials)

[0288] 1. Experimental Objective

[0289] This study aims to verify the gel-forming ability and physical stability of the recombinant type III collagen (Group2) in this application relative to wild-type human type III collagen (Group3) and blank control (Group1), evaluate its basic performance as a biological scaffold material, and demonstrate its advantages in high activity and stability.

[0290] 2. Experimental Materials

[0291] 2.1 Strains and Sample Preparation

[0292] Group 1 sample: Pichia pastoris strain GS115 without exogenous gene transfer was cultured for 72 h using the shake-flask induction method in Example 3. The fermentation supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization (it does not contain collagen and serves as a negative control).

[0293] Group 2 samples: Recombinant Pichia pastoris strain expressing recombinant type III collagen (SEQ ID NO.3) was purified, dissolved in 0.01 M acetic acid solution, and the concentration was adjusted to 2 mg / mL (purity ≥95% verified by BCA quantification).

[0294] Group 3 sample: Recombinant Pichia pastoris strain expressing wild-type human type III collagen (SEQ ID NO.2) was purified using the above method and the concentration was adjusted to 2 mg / mL.

[0295] 2.2 Reagents and Instruments

[0296] Reagents: 10×PBS (pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), deionized water (conductivity ≤ 1.0 μS / cm), 0.01 M acetic acid solution (for dissolving collagen).

[0297] Instruments: rheometer, 37℃ constant temperature water bath, analytical balance (accuracy 0.1 mg).

[0298] 3 Experimental Steps

[0299] 3.1 Gel preparation and visualization verification

[0300] Prepare the mixture according to the volume ratio of "collagen solution: 10×PBS: deionized water = 8:1:1": Take 800 μL of 2 mg / mL collagen solution for each group (800 μL of Group 1 supernatant is used instead for Group 1), add 100 μL of 10×PBS and 100 μL of deionized water, gently invert to mix (avoid air bubbles), and immediately transfer to a 5 mL centrifuge tube.

[0301] Six parallel samples were set up for each group, and the experiment was repeated three times. The centrifuge tubes were placed vertically in a 37°C water bath and removed at 5 min, 10 min, 15 min, 20 min and 30 min respectively. They were then inverted for 10 s to observe whether they flowed (complete non-flow indicates gel formation) and the time for complete gelation was recorded.

[0302] 3.2 Rheological property testing

[0303] Take 1 mL of fresh gel (after 30 min of complete formation) and place it on the sample stage of the rheometer. Adjust the spacing between the parallel plates to 1 mm. Remove excess gel with a scraper. Set the parameters as follows: temperature 37℃, frequency 1 Hz (simulating the vibration frequency of the physiological environment), strain 0.1% (linear viscoelastic region, to avoid damaging the gel structure). Continuously detect for 30 min, and record the storage modulus (G', Pa) and loss modulus (G, Pa) every 30 s.

[0304] Calculate the G' / G” ratio (reflecting the proportion of gel elasticity), and take the mean of 6 parallel samples for each group.

[0305] 3.3 Storage stability test

[0306] Seal the remaining gel (5 mL centrifuge tube) and refrigerate at 4°C. Observe its appearance daily (whether it separates into layers, breaks, or separates into water) and record the phenomena.

[0307] On day 7, the rheological test in section 3.2 was repeated, and the rate of change of G' was calculated as: (initial G' - 7d G') / initial G' × 100%.

[0308] Statistical methods: One-way ANOVA was performed using GraphPad Prism 9. LSD-t test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0309] 4. The experimental results are shown in Table 6.

[0310] Table 6

[0311]

[0312] 5. Results Analysis and Conclusions

[0313] (1) Verification of gel-forming ability:

[0314] Group 1 failed to form a gel (it remained fluid even after being inverted for 30 minutes), proving that the host strain's own components could not form a gel structure.

[0315] The complete gelation time of Group 2 (18.5 min) was significantly shorter than that of Group 3 (25.3 min, P<0.01), indicating that the gelation efficiency of recombinant collagen was higher.

[0316] (2) Stability of gel structure:

[0317] The G' of fresh gels was significantly higher than that of G (Group 2 G' / G" = 2.00 > 1.5, Group 3 = 1.84 > 1.5), which met the criteria for stable gels;

[0318] The G' (1580 Pa) of Group 2 was significantly higher than that of Group 3 (1250 Pa, P<0.01), indicating that its gel has stronger elasticity and a denser structure.

[0319] (3) Storage stability:

[0320] After storage at 4℃ for 7 days, Group 2 showed no water separation, stratification, or fragmentation, while Group 3 showed slight water separation (<5% by volume).

[0321] The rate of change of G' in Group 2 (5.2%) was significantly lower than that in Group 3 (8.5%, P<0.05), and both were ≤10%, demonstrating that both groups met the requirements for biomaterial storage, and that the recombinant collagen had better stability.

[0322] 6. Data reliability

[0323] Each group has 6 parallel samples, the experiment is repeated 3 times, and the data are presented as "mean ± standard deviation" to reduce operational errors;

[0324] Rheological testing was conducted under strict temperature (37°C) and strain (0.1%) conditions to ensure that gel properties were evaluated under physiologically relevant conditions.

[0325] Statistical analysis showed that the difference between Group 2 and Group 3 was statistically significant (P<0.05 or P<0.01), which fully demonstrates that the gel-forming ability and stability of recombinant type III collagen are significantly better than those of wild type, meeting the core requirements of biological scaffold materials.

[0326] This result is consistent with the proliferation and migration results, further verifying the high activity and practicality of the recombinant type III collagen in this application from the perspective of the physical properties of biomaterials.

[0327] Experiment 4: Thermal stability test of recombinant type III collagen

[0328] 1. Experimental Objective

[0329] The thermal denaturation temperature (Tm) of the recombinant type III collagen (Group2) of this application relative to wild-type human type III collagen (Group3) and blank control (Group1) was verified, and its stability in storage (4°C) and application (37°C) environments was evaluated to demonstrate its thermal stability advantage and application safety.

[0330] 2. Experimental Materials

[0331] 2.1 Strains and Sample Preparation

[0332] Group 1 sample: Pichia pastoris strain GS115 without exogenous gene transfer was cultured for 72 h using the shake-flask induction method in Example 3. The fermentation supernatant was collected, filtered through a 0.22 μm filter membrane for sterilization, and diluted with PBS (pH 7.4) to the same volume as the experimental group (without collagen, as a negative control).

[0333] Group 2 samples: Recombinant Pichia pastoris strain expressing recombinant type III collagen (SEQ ID NO.3) was taken, purified and dissolved in PBS (pH 7.4), and the concentration was adjusted to 1 mg / mL (BCA quantification verified purity ≥95%, no protein aggregation).

[0334] Group 3 sample: Recombinant Pichia pastoris strain expressing wild-type human type III collagen (SEQ ID NO.2) was purified using the method described above and the concentration was adjusted to 1 mg / mL (dissolved in PBS, pH 7.4).

[0335] 2.2 Reagents and Instruments

[0336] Reagents: PBS buffer (pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, sterilized by filtration through a 0.22 μm filter membrane), ultrapure water (conductivity ≤ 1.0 μS / cm).

[0337] Instruments: Circular dichroism chromatograph (JASCO J-815, equipped with temperature control system), quartz cuvette (1 mm optical path, quartz material, no UV absorption), sample degassing device, analytical balance (accuracy 0.1 mg).

[0338] 3. Experimental Procedure

[0339] 3.1 Sample Pretreatment

[0340] Take 200 μL of each sample (Group1 / Group2 / Group3) and place it in a quartz cuvette. Use PBS as a blank control (same sample volume) and equilibrate at 25℃ for 30 min.

[0341] The samples were degassed (to avoid generating bubbles that would interfere with the signal during scanning), and three parallel samples were set up for each group. The experiment was repeated three times.

[0342] 3.2 Circular Dichroism Scanning Parameter Settings

[0343] Wavelength range: 190-250 nm (characteristic absorption range of collagen triple helix structure, with 222 nm as the characteristic absorption peak).

[0344] Temperature scan range: 25℃-95℃, heating rate 1℃ / min;

[0345] Scan bandwidth: 1 nm, response time: 1 s, scan speed: 50 nm / min;

[0346] Data acquisition: The molar ellipticity ([θ]) at 222 nm was recorded once for every 1°C increase in temperature. 222Unit: deg cm 2 dmol -1 At the same time, the full-wavelength scan spectrum was recorded (to verify the triple helix structure characteristics).

[0347] 3.3 Tm value calculation and data processing

[0348] Molar ellipticity ([θ]) at 222 nm, plotted with temperature on the x-axis. 222 Plot the thermal deformation curve with y as the ordinate;

[0349] Determine the curve baseline: use [θ] at 25℃. 222 As the "undenatured baseline," [θ] at 95℃ is used. 222 As a “completely altered baseline”;

[0350] Calculate the Tm value: Locate [θ] in the thermal denaturation curve. 222 The temperature at which the temperature drops to 50% of the difference between the undenatured baseline and the fully denatured baseline is the Tm value (the midpoint temperature at which the triple helix structure unwinds).

[0351] Statistical methods: One-way ANOVA was performed using GraphPad Prism 9. LSD-t test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0352] 4. The experimental results are shown in Table 7.

[0353] Table 7

[0354]

[0355] Note: [θ] 222 Retention rate = (at a certain temperature [θ]) 222 Absolute value / at 25℃ [θ] 222 (Absolute value) × 100%, the higher the retention rate, the more complete the triple helix structure.

[0356] 5. Results Analysis and Conclusions

[0357] (1) Verification of the triple helix structure:

[0358] Group 1 showed no significant characteristic absorption peaks in the 190-250 nm range (close to PBS blank), proving that the host strain did not interfere.

[0359] Both Group 2 and Group 3 showed a significant negative absorption peak at 222 nm (Group 2: -13800deg). cm 2 dmol-1 Group 3: -12500deg cm 2 dmol -1 Furthermore, the absorption peak intensity of Group 2 was significantly higher than that of Group 3 (P<0.05), demonstrating that the triple helix structure of recombinant collagen is more complete (the basis of active structure).

[0360] (2) Comparison of core thermal stability indicators (Tm value):

[0361] The Tm value for Group 3 was 41.3℃;

[0362] The Tm value of Group 2 was 48.5℃, which was significantly higher than that of Group 3 (P<0.01), and was ≥45℃, meeting the criterion of "thermal stability is better than that of natural protein";

[0363] Both sets of Tm values ​​were ≥37℃, proving that the cells would not be deactivated in the application environment.

[0364] (3) Stability verification in actual application scenarios:

[0365] At 37℃ (body surface application temperature), [θ] of Group 2 222 The retention rate reached 97.8%, which was significantly higher than that of Group 3 (92.5%, P<0.05), proving that its triple helix structure is not easy to unwind during application;

[0366] At 45℃ (extreme storage / transport temperature), the retention rate of Group2 was still 90.1%, while that of Group3 was only 78.3% (P<0.01), indicating that recombinant collagen is more resistant to temperature fluctuations and has higher safety for long-term storage (4℃) and transportation.

[0367] 6. Data reliability

[0368] Each group has 3 parallel samples, and the experiment is repeated 3 times. The data are presented as "mean ± standard deviation" to reduce instrument error.

[0369] The circular dichroism chromatographic scanning parameters strictly follow the industry standards for collagen thermal stability testing (heating rate 1℃ / min, characteristic wavelength 222nm) to ensure comparable results;

[0370] Statistical analysis shows the T-values ​​and [θ] of Group2 and Group3. 222 The differences in retention rates were all statistically significant (P<0.05 or P<0.01), fully demonstrating that the thermal stability of recombinant type III collagen is significantly better than that of wild type, meeting the safety requirements for storage and application.

[0371] Experiment 5: Cell adhesion ability test of recombinant type III collagen

[0372] 1. Experimental Objective

[0373] This study aims to verify the adhesion-promoting effect of the recombinant type III collagen (Group2) of this application relative to wild-type human type III collagen (Group3) and blank control (Group1) on wound repair-related cells (human skin fibroblasts HSF and human umbilical vein endothelial cells HUVEC), assess its biocompatibility, and demonstrate its fundamental advantages as a biomaterial for cell interaction.

[0374] 2. Experimental Materials

[0375] 2.1 Strains and Sample Preparation

[0376] Group 1 sample: Pichia pastoris strain GS115 without exogenous gene transfer was cultured for 72 h using the shake-flask induction method in Example 3. The fermentation supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization (it does not contain collagen and serves as a negative control).

[0377] Group 2 samples: Recombinant Pichia pastoris strain expressing recombinant type III collagen (SEQ ID NO.3) was purified, dissolved in PBS (pH 7.4), and serially diluted to 1 μg / mL, 5 μg / mL, and 10 μg / mL (BCA quantification verified purity ≥95%).

[0378] Group 3 samples: Recombinant Pichia pastoris strain expressing wild-type human type III collagen (SEQ ID NO.2) was purified and serially diluted to 1 μg / mL, 5 μg / mL, and 10 μg / mL (dissolved in PBS, pH 7.4) using the same method as described above.

[0379] 2.2 Cells and Reagents

[0380] Cells: Human skin fibroblasts (HSF, ATCC number CRL-2522) and human umbilical vein endothelial cells (HUVEC, ATCC number CRL-1730) were cultured to the 5th generation for experiments.

[0381] Reagents: DMEM medium containing 10% FBS, serum-free DMEM medium, PBS buffer (pH 7.4), 4% paraformaldehyde solution (prepared with PBS, freshly prepared), 0.1% crystal violet staining solution (dissolved in deionized water and filtered for sterilization), 33% glacial acetic acid solution (analytical grade), and 1% BSA blocking solution (dissolved in PBS).

[0382] Consumables: 96-well cell culture plate, sterile pipette tips.

[0383] 2.3 Instruments

[0384] Inverted microscope (Olympus, 100× magnification), microplate reader (BioTek, detection wavelength 570 nm), CO2 incubator (Thermo, 37℃, 5% CO2), centrifuge (800 rpm), analytical balance (accuracy 0.1 mg).

[0385] 3. Experimental Procedure

[0386] 3.1 96-well plate coating and sealing

[0387] Coating: Add 100 μL / well of collagen samples of different concentrations (1 / 5 / 10 μg / mL concentration groups of Group 2 and Group 3) to 96-well plates, add 100 μL / well of fermentation supernatant to Group 1, and set up a blank coating group (PBS only). Incubate overnight at 4°C (12-16 h).

[0388] Wash plate: Discard the coating solution, gently rinse twice with PBS (150 μL / well each time to avoid excessive impact that could damage the coating layer), and shake off any remaining liquid.

[0389] Blocking: Add 150 μL of 1% BSA blocking solution to each well, incubate at 37°C for 1 h (to block nonspecific adhesion), discard the blocking solution, rinse twice with PBS, and spin dry for later use.

[0390] 3.2 Cell Seeding and Adhesion Culture

[0391] Cell pretreatment: HSF and HUVECs were resuspended in DMEM medium containing 10% FBS and the cell density was adjusted to 1×10⁶ cells / year. 4 Cells / 100 μL, starved with serum-free DMEM for 1 h (synchronizing cell state).

[0392] Inoculation: HSF and HUVEC were inoculated into the coated 96-well plate at 100 μL per well, with 6 replicates for each concentration in each group, and the experiment was repeated 3 times.

[0393] Adhesion culture: Incubate statically at 37℃ and 5% CO2 for 1 h (short-term adhesion to avoid cell proliferation interfering with the results).

[0394] 3.3 Removal and Fixation of Non-Adhering Cells

[0395] Wash away unattached cells: Gently remove the 96-well plate and slowly add 150 μL of PBS per well along the well wall. Tilt the plate to discard the liquid and repeat twice (to ensure the removal of free cells).

[0396] Fixation: Add 100 μL of 4% paraformaldehyde solution to each well and fix at room temperature for 15 min. Discard the fixative and rinse once with PBS.

[0397] Staining: Add 100 μL of 0.1% crystal violet staining solution to each well, stain at room temperature in the dark for 20 min, discard the staining solution, rinse slowly with deionized water 3 times (until the rinsing solution is colorless), and air dry upside down.

[0398] 3.4 Detection and Data Calculation

[0399] Dye dissolution: Add 100 μL of 33% glacial acetic acid solution to each well and shake horizontally for 10 min to completely dissolve the crystal violet.

[0400] Absorbance detection: The OD value at 570 nm was measured using an ELISA reader, and the data was recorded.

[0401] Adhesion rate calculation: Adhesion rate (%) = [(OD value of experimental group - OD value of blank coated group) / (OD value of blank coated group)] × 100%.

[0402] Statistical methods: Two-way ANOVA (cell type × sample group) was performed using GraphPad Prism 9. LSD-t test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0403] 4. The experimental results are shown in Table 8.

[0404] Table 8

[0405]

[0406] 5. Results Analysis and Conclusions

[0407] Blank control interference exclusion: The adhesion rate of HSF and HUVEC in Group 1 (GS115 supernatant) was not significantly different from that in the blank coated group (P>0.05), proving that the components of the host strain itself do not affect cell adhesion, and the experimental results are directly mediated by collagen.

[0408] (1) Adhesion activity comparison (Group2 vs Group3):

[0409] The adhesion rate of both cell types increased with increasing collagen concentration, and the adhesion rate of the 5-10 μg / mL group was significantly higher than that of the 1 μg / mL group (P<0.05).

[0410] Group 2 showed significantly higher adhesion rates at all concentrations than Group 3 at the same concentration (P<0.05 or P<0.01), with the 10 μg / mL concentration group showing the most significant advantage: HSF adhesion rate reached 232.6% (Group 3 was 166.0%), and HUVEC adhesion rate reached 239.0% (Group 3 was 160.6%), both far exceeding the excellent standard of ≥60%.

[0411] (2) Biocompatibility verification:

[0412] Group 2 showed strong adhesion-promoting effects on both HSF and HUVEC, with no cell-specific preference, demonstrating good compatibility with different types of wound repair cells (skin fibroblasts, vascular endothelial cells).

[0413] Microscopic observation showed that the adhered cells in Group 2 spread more fully and had regular morphology (HSF was spindle-shaped and HUVEC was polygonal), with obvious pseudopodia extension, while the cells in Group 3 spread less.

[0414] Optimal concentration determination: 10 μg / mL is the optimal cell adhesion concentration for Group 2. This concentration is consistent with the optimal concentration for proliferation and migration, providing a unified parameter support for subsequent applications.

[0415] 6. Data reliability

[0416] Each group was set up with 6 replicates, and the two types of cells were tested simultaneously. The experiment was repeated 3 times, and the data were presented as "mean ± standard deviation" to reduce systematic error.

[0417] Strictly control the adhesion time (1 h) to avoid cell proliferation interfering with the adhesion results; standardize the staining and washing steps to ensure the accuracy of OD value detection.

[0418] Statistical analysis showed that the differences between groups were statistically significant (P<0.05 or P<0.01), which fully demonstrates that the cell adhesion activity of recombinant type III collagen (Group2) is significantly better than that of wild type (Group3), and that it has better biocompatibility, meeting the core application requirements of biomaterials.

[0419] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

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

3.

2. A biomaterial, characterized in that, The biomaterial includes any one of the following A1)-A5): A1) A nucleic acid molecule, said nucleic acid molecule encoding the recombinant type III collagen of claim 1; A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

3. The biomaterial according to claim 2, characterized in that, The nucleic acid molecule is shown in SEQ ID NO.

1.

4. The biomaterial according to claim 2, characterized in that, The recombinant microorganisms are selected from one or more of Streptococcus, Bacillus, Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.

5. A method for preparing recombinant type III collagen, characterized in that, The method includes: constructing a recombinant microorganism expressing the recombinant type III collagen as described in claim 1, and culturing the recombinant microorganism.

6. The preparation method according to claim 5, characterized in that, The culture conditions are: temperature 24℃-30℃, pH value 5-6.

7. The use of the recombinant type III collagen according to claim 1 or the recombinant type III collagen prepared by the preparation method according to claim 5 or 6 in any one or more of the following B1)-B7): B1) Prepare products that promote cell proliferation; B2) Prepare products that promote cell migration; B3) Preparation of wound repair products; B4) Promotes gel formation; B5) Preparation of biological scaffold-related products; B6) Improve the storage stability and / or thermal stability of collagen products; B7) Prepare products that improve cell adhesion properties.

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