Type IV recombinant collagen and preparation method and application thereof

Abstract

The application discloses IV type recombinant collagen protein and a preparation method and application thereof, and relates to the technical field of genetic engineering. The recombinant collagen protein comprises a recombinant collagen single chain; wherein the recombinant collagen single chain is obtained by repeating a collagen monomer fragment as shown in SEQ ID NO. 2 for 2-10 times. The application designs and obtains a recombinant collagen amino acid sequence which can be stably and efficiently expressed in Pichia pastoris and has excellent biological activity by accurately screening and optimizing the functional region sequence of human natural IV type collagen. The sequence is 100% homologous to the corresponding region of human natural IV type collagen, thereby ensuring low immunogenicity of the product and excellent biocompatibility with the human body from the source.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a type IV recombinant collagen, its preparation method, and its applications. Background Technology

[0002] Type IV collagen is a key component of the human basement membrane network structure, playing an irreplaceable role in maintaining tissue structure, mediating cell adhesion and migration, and regulating tissue repair and regeneration. Compared to fibrous collagens such as Type I and Type III, the unique network structure of Type IV collagen endows it with superior bioactivity, making it a promising candidate for applications in high-end medical devices, tissue engineering scaffolds, and functional biomaterials. However, traditional methods, which rely heavily on animal tissue extraction, are inherently flawed, facing risks such as viral contamination, significant batch-to-batch variations, potential immunogenicity, and ethical controversies. Therefore, developing high-purity, highly active, and precisely sequenced humanized Type IV collagen using gene recombination technology has become a core direction for the development of biomedical materials.

[0003] Chinese patent CN114940712A discloses a method for preparing biosynthetic human structural materials. It employs an *E. coli* prokaryotic expression system, screening functional region sequences of natural human type IV collagen for tandem repeats, and then expressing the target protein intracellularly. However, the prokaryotic system lacks the post-translational modification capabilities unique to eukaryotes, resulting in differences in the spatial conformation of the recombinant protein compared to the natural human protein, thus limiting its biological activity. Furthermore, intracellular expression requires a cell disruption step, and if inclusion bodies are formed, in vitro refolding is necessary, making the process cumbersome and yielding low results. Finally, the *E. coli* system carries a high risk of endotoxin residue, and subsequent removal processes increase production costs and control difficulties, posing a challenge for products with high safety requirements, such as injectable medical devices.

[0004] Chinese patent CN117801095A employs a Pichia pastoris eukaryotic secretory expression system, introducing non-natural linker peptides that can be specifically cleaved by endogenous host proteases between monomeric repeat sequences, aiming to achieve automatic removal of linker peptides during secretion. However, its reliance on endogenous enzymatic cleavage mechanisms makes it difficult to guarantee product uniformity—cleavage efficiency is significantly affected by fermentation conditions; incomplete cleavage leaves non-natural amino acid sequences in the final product, posing a potential immunogenicity risk; excessive cleavage may damage the integrity of the target domain; simultaneously, the mandatory linker peptide design limits the flexibility of sequence design, making it difficult to adapt to different types of type IV collagen functional fragments; under high-density fermentation, excessive secretion of exogenous proteins easily triggers unfolded protein reactions, leading to target protein degradation or misfolding, making it difficult to achieve both high expression and high activity.

[0005] Therefore, there is an urgent need to develop a novel method for preparing recombinant type IV collagen that can be directly and efficiently expressed, has a stable structure, excellent activity, and does not require exogenous enzyme digestion or residual linker peptides, in order to meet the urgent needs of large-scale production and high-end biomedical applications. Summary of the Invention

[0006] Therefore, the present invention provides a type IV recombinant collagen, its preparation method and application, to solve the problems in the prior art.

[0007] This invention selects some genes of natural collagen in the human body, performs certain optimization combinations, and designs a stable and highly bioactive type IV collagen amino acid sequence.

[0008] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a type IV recombinant collagen comprises a recombinant collagen single chain; wherein the recombinant collagen single chain is obtained by repeating a collagen monomer fragment as shown in SEQ ID NO.2 2 to 10 times. Preferably, it is repeated 3 to 7 times.

[0009] SEQ ID NO. 2: GDPGLKGDKGDVGLPGKPGSMGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGPQGSPGLPGDKGAKGEKGQAGPP This invention allows for precise control of the number of repetitions, enabling flexible adjustment of the molecular weight of recombinant proteins. Fewer repetitions (2-3 times) facilitate rapid protein penetration, making it suitable for transdermal absorption products such as cosmetic serums; more repetitions (4-7 times) create a more robust network structure, providing excellent mechanical support and moisturizing film-forming properties, making it particularly suitable for medical devices (such as hemostatic sponges and tissue-engineered scaffolds) and long-acting sustained-release drug carriers. This controllability solves the problem of fixed molecular weight and difficulty in optimizing for different application scenarios in existing technologies.

[0010] Furthermore, the recombinant collagen monomer fragment is obtained by cutting and splicing the amino acid sequence of human type IV collagen; wherein the amino acid sequence of human type IV collagen is shown in SEQ ID NO. 1.

[0011] SEQ ID NO. 1: Furthermore, the recombinant collagen monomer fragment is obtained by sequentially combining amino acids at positions 918-938, 1115-1147, and 1032-1055 of human type IV collagen. This invention, through in-depth analysis of the full-length sequence of human type IV collagen, specifically selected three functional fragments at positions 918-938, 1115-1147, and 1032-1055 to form a monomer fragment. This "three-segment" combination design cleverly preserves the key amino acid motifs in type IV collagen responsible for cell adhesion, recognition, and triple-helix structure stability. Subsequent experiments showed that the monomer formed by this specific combination, after repeated construction, can more effectively mimic the network structure characteristics of natural type IV collagen, significantly improve the protein's thermal stability and resistance to enzymatic degradation, and greatly enhance its adhesion and migration-promoting effects on fibroblasts and keratinocytes, exhibiting superior repair and regeneration activity compared to existing technologies.

[0012] This invention does not rely on any endogenous or exogenous protease cleavage, fundamentally avoiding the problem of non-natural sequence residues caused by incomplete enzymatic cleavage, and ensuring the accuracy and uniformity of the final product sequence; the product does not contain any non-human amino acid sequences, and will not cause an immune response due to the introduction of exogenous sequences when used in humans.

[0013] Furthermore, the nucleotide sequence encoding the collagen is shown in SEQ ID NO.4.

[0014] SEQ ID NO.4: ggcgatccaggcctcaaaggcgataaaggcgatgtcgggcttcccggtaagccggggtcgatgggtgaaaaaggagataaaggtctacccgggcttgacggtattcccggagtgaagggggaagctgggctcccgggcacgcctggaccaacagggccagccgggcctcaaggttcaccagggctgcctggagacaagggagcgaaaggggagaagggccaggcaggaccgccgggtgatccagggttaaagggtgataagggagatgtgggtttaccgggaaaacccgggtctatgggggagaagggggacaagggattgcccggcttagatggtattcctggagtaaagggtgaagcagggctacctgggacgcccgggccgactggtcctgcgggcccacagggttctcccgggctgcctggggacaaaggtgccaaaggtgaaaaaggacaagcgggaccaccaggcgatccgggtcttaaaggagataagggtgacgtcggtctcccggggaagcctggaagtatgggagagaaaggcgacaaagggttgccaggtcttgacggcatacccggcgtaaagggagaagcaggcctgccgggcacccctggacctaccggcccagctggtcctcaaggctcacccggactaccgggcgacaaaggggcaaaaggggaaaagggtcaggctggccctcctggagatcccggactcaagggagataaaggggacgttggtctgccgggaaagcccgggagcatgggcgagaaaggtgacaagggattacccggcttggacggcatacctggtgttaaaggcgaggccgggctaccgggaacacccggcccaactggaccagcgggccctcaaggttctcctggcttgccgggtgacaagggtgctaagggtgagaaaggtcaggccggcccgccc A method for preparing a type IV recombinant collagen according to the second aspect of the present invention, the method comprising: Step 1: Design collagen sequences The amino acid sequences of human type IV collagen, as shown in SEQ ID NO. 1, are sequentially taken from positions 918-938, 1115-1147, and 1032-1055 to obtain recombinant collagen monomer fragments; this process is repeated 2 to 10 times to obtain recombinant collagen single chains. Step 2, constructing the carrier Based on the complete nucleotide sequence of the recombinant collagen single strand, it is recombined into a plasmid to obtain a recombinant plasmid; Step 3: Transformation and Cultivation The recombinant plasmid was transferred into a eukaryotic host cell, and engineered strains were screened. The engineered strains were then fermented and cultured to induce the expression of the target protein under appropriate conditions. Step 4: Harvesting and Purification The fermentation broth was collected, the expressed protein product was harvested, and the protein product was purified to obtain high-purity target collagen.

[0015] Furthermore, in step two, the plasmid is selected for prokaryotic expression, specifically the pET series vector, pGEX series vector, pBAD series vector, or pUC series vector. As an example, preferred plasmids include pET-28a, pET-32a, pGEX-4T-1, pBAD / Myc-His, and pUC19.

[0016] Furthermore, in step two, the plasmid is selected for eukaryotic expression, specifically pcDNA series vectors, pCMV series vectors, pIRES series vectors, pEGFP series vectors, pYES series vectors, or pPIC series vectors. As examples, pcDNA3.1, pCMV-Tag, pCMV-Sport, pIRES2-EGFP, pEGFP-C1, pYES2, pPICZ, and pPIC3.5 are preferred.

[0017] Furthermore, in step three, the host cell is a yeast cell or a mammalian cell. As an example, yeast cells are preferred, specifically Pichia pastoris or Saccharomyces cerevisiae; mammalian cells are preferred, specifically HEK293 cells or CHO cells.

[0018] This invention preferably employs a Pichia pastoris eukaryotic expression system, directly secreting the target protein into the fermentation supernatant. The eukaryotic system possesses post-translational modification capabilities such as proline and lysine hydroxylation, which helps to form a triple helix structure closer to the native conformation, ensuring biological activity. The secretory expression pathway guides the protein to fold correctly in the endoplasmic reticulum, forming a functional conformation before secretion into the extracellular space, avoiding the inclusion bodies and subsequent refolding problems that may occur with intracellular expression. The endotoxin level of the yeast system is much lower than that of Gram-negative bacteria Escherichia coli, resulting in higher product safety, making it particularly suitable for high-requirement scenarios such as injectable medical devices.

[0019] The third aspect of the present invention provides the application of a type IV recombinant collagen in the preparation of collagen-containing products.

[0020] Furthermore, the collagen products include cosmetics, medical devices, or pharmaceuticals.

[0021] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers; Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials; Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0022] In one embodiment, the medicine includes, but is not limited to, wound healing materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (for rapid hemostasis), and collagen membrane (for postoperative repair). Cartilage repair materials, such as collagen injections (for repairing articular cartilage defects) and collagen-based cartilage grafts; Drug delivery carriers, such as collagen microspheres (encapsulating active ingredients for sustained drug release) and collagen gel injections (for local delivery of anti-inflammatory or anti-tumor drugs).

[0023] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds (for cell culture and tissue regeneration), collagen-based composite materials (for bone or soft tissue repair), and collagen filler materials (for filling tissue or cavities). Surgical aids include, but are not limited to, collagen sutures (biodegradable sutures) and collagen hemostatic sponges (for intraoperative hemostasis). Oral restoration materials include, but are not limited to, collagen membranes (alveolar bone filling) and collagen periodontal regeneration materials.

[0024] In one embodiment, cosmetics include skincare products, such as collagen masks (moisturizing, repairing, and anti-aging), collagen serums (improving skin elasticity), and collagen lotions (hydrating and locking in moisture). Hair care products, such as collagen shampoo (to repair damaged hair) and collagen conditioner (to replenish nutrients and make hair smooth). Anti-aging products, such as collagen anti-wrinkle cream (to improve skin firmness) and collagen freeze-dried powder (to promote skin regeneration and repair fine lines).

[0025] A fourth object of the present invention is to provide a collagen product containing any of the above-described recombinant collagen single chains; Optionally, the collagen product may be a pharmaceutical, medical device, or cosmetic.

[0026] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers; Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials; Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0027] In one embodiment, the medicine includes, but is not limited to, wound healing materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (for rapid hemostasis), and collagen membrane (for postoperative repair). Cartilage repair materials, such as collagen injections (for repairing articular cartilage defects) and collagen-based cartilage grafts; Drug delivery carriers, such as collagen microspheres (encapsulating active ingredients for sustained drug release) and collagen gel injections (for local delivery of anti-inflammatory or anti-tumor drugs).

[0028] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds (for cell culture and tissue regeneration), collagen-based composite materials (for bone or soft tissue repair), and collagen filler materials (for filling tissue or cavities). Surgical aids include, but are not limited to, collagen sutures (biodegradable sutures) and collagen hemostatic sponges (for intraoperative hemostasis). Oral restoration materials include, but are not limited to, collagen membranes (alveolar bone filling) and collagen periodontal regeneration materials.

[0029] In one embodiment, cosmetics include skincare products, such as collagen masks (moisturizing, repairing, and anti-aging), collagen serums (improving skin elasticity), and collagen lotions (hydrating and locking in moisture). Hair care products, such as collagen shampoo (to repair damaged hair) and collagen conditioner (to replenish nutrients and make hair smooth). Anti-aging products, such as collagen anti-wrinkle cream (to improve skin firmness) and collagen freeze-dried powder (to promote skin regeneration and repair fine lines).

[0030] The present invention has the following advantages: This invention designs a recombinant collagen amino acid sequence that can be stably and efficiently expressed in Pichia pastoris and exhibits excellent biological activity by precisely screening and optimizing the functional region sequence of natural human type IV collagen. This sequence is 100% homologous to the corresponding region of natural human type IV collagen, ensuring low immunogenicity and excellent biocompatibility of the product from the source.

[0031] The recombinant collagen prepared by this invention exhibits outstanding structural stability: after heating at 60°C for 4 hours, SDS-PAGE analysis showed no significant changes in the protein bands, indicating that the structure and purity remained intact. Furthermore, the protein maintained good stability within a pH range of 6.0–8.0, demonstrating broad pH adaptability. This property gives it significant advantages in complex processing conditions and various clinical applications.

[0032] The sequence design of this invention eliminates the dependence on exogenous linker peptides and endogenous host enzyme cleavage, thus fundamentally avoiding the risk of protein degradation. In high-density Pichia pastoris fermentation at a scale of 5L fermenter, the target protein showed a single band in the fermentation supernatant, with the main band accounting for over 80%, and no obvious degradation bands were observed. This excellent expression stability ensures batch-to-batch consistency of the product, significantly reduces purification difficulty and cost, and possesses good potential for industrial production.

[0033] Cellular functional verification showed that the recombinant collagen prepared in this invention has excellent biological functions.

[0034] This invention achieves a synergistic balance of high stability, high expression activity, high bioactivity, and high safety while ensuring precise product sequence and extremely low immunogenicity. It overcomes the technical bottleneck of prior art where "expression stability" and "bioactivity" are difficult to balance, and provides a better solution for the large-scale application of recombinant type IV collagen in high-end medical devices, tissue engineering, and functional skin care products. Attached Figure Description

[0035] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0036] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0037] Figure 1 This is a schematic diagram of the recombinant plasmid provided in Embodiment 1 of the present invention; Figure 2 The images show the results of recombinant collagen from Pichia pastoris genetically engineered bacteria provided in Example 1 of this invention; wherein, 2A is the SDS-PAGE detection result of the supernatant from shake-flask expression; and 2B is the SDS-PAGE detection result of the supernatant from 5L large-scale fermentation. Figure 3 This is a graph showing the thermal stability results of recombinant collagen COL4-4 ​​provided in Test Example 1 of the present invention; Figure 4 The test results of the acid and alkali resistance of recombinant collagen COL4-4 ​​provided in Test Example 1 of this invention; Figure 5 This is a graph showing the results of the recombinant collagen COL4-4 ​​cell adhesion activity assay provided in Test Example 1 of this invention. Figure 6 This is a graph showing the detection results of the recombinant collagen COL4-4 ​​cell proliferation activity provided in Test Example 1 of the present invention; Figure 7 This is a graph showing the cytotoxicity test results of recombinant collagen COL4-4 ​​provided in Test Example 1 of the present invention; Figure 8 The image shows the evaluation results of diabetic wound healing provided in Test Example 2 of this invention. Detailed Implementation

[0038] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0040] Seed culture medium YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose); Fermentation medium (glycerol 46 g / L, magnesium sulfate heptahydrate 2.02 g / L, calcium sulfate dihydrate 0.46 g / L, potassium sulfate 9.1 g / L, phosphate 11.34 ml / L, potassium hydroxide 2.06 g / L, PTM1 trace element 4 ml / L); fed-batch medium (50% w / v glycerol, with 12 ml / L PTM1 trace element per liter). Induction medium (100% methanol, with 12 mL of PTM1 trace elements added per liter); PTM1: sterilized by filtration through a 0.22 μm filter membrane and stored at 4°C. After the fermentation medium is sterilized at high temperature, PTM1 is added after the temperature drops to room temperature, and the pH is adjusted to 5 with ammonia. pPICzα empty vector was purchased from Thermo Fisher Scientific. SacI was acquired from Thermo Fisher.

[0041] Example 1 This embodiment provides a method for preparing type IV recombinant collagen: 1. Design collagen sequences Based on the sequence of human type IV collagen (https: / / www.uniprot.org / uniprot / P02462; COL4A1, amino acid sequence as shown in SEQ ID NO.1), recombinant collagen was designed as follows: The amino acid sequences of positions 918-938, 1115-1147, and 1032-1055 were sequentially taken to obtain recombinant collagen monomer fragment 1, with the amino acid sequence shown in SEQ ID NO.2. SEQ ID NO.2 was repeated 4 times to obtain recombinant collagen single chains, named 1-4, with the amino acid sequences shown in SEQ ID NO.3.

[0042] 2. Construction of recombinant plasmids and recombinant bacteria The amino acid sequence SEQ ID NO.3 obtained in step 1 was used to complete the corresponding nucleotide sequence (corresponding to nucleotide sequence SEQ ID NO.4) by Nanjing Genscript Biotech Co., Ltd., and inserted into the α-factor reading frame of the pPICzα empty vector (purchased from Thermo Fisher Scientific) to obtain the recombinant plasmid, named pPICzα-COL4-4 ​​(also known as COL4-1_pPICZalphaA) (amino acid sequence SEQ ID NO.3, nucleotide sequence SEQ ID NO.4). A schematic diagram of the plasmid is shown below. Figure 1 As shown.

[0043] SEQ ID NO.3: GDPGLKGDKGDVGLPGKPGSMGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGPQGSPGLPGDKGAKGEKGQAGPP GDPGLKGDKGDVGLPGKPGSMGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGPQGSPGLPGDKGAKGEKGQAGPP GDPGLKGDKGDVGLPGKPGSMGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGPQGSPGLPGDKGAKGEKGQAGPP GDPGLKGDKGDVGLPGKPGSMGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGPQGSPGLPGDKGAKGEKGQAGPP The above recombinant plasmid (2-3 μg) was digested with SacI (purchased from Thermo Fisher) at 37℃ for 2 h, linearized, recovered, and electroporated into Pichia pastoris X33 competent cells; the electroporated bacterial culture was plated on YPD / Zeocin plates and incubated upside down at 30℃ for 2-5 days. Single colonies were selected to verify the Pichia pastoris genetic engineering, which was named P. pastoris-COL4-4.

[0044] 3. Induced expression and identification of recombinant collagen The Pichia pastoris genetically engineered strain obtained in step 2 was inoculated into 50 mL centrifuge tubes containing 5 mL of BMGY medium and cultured at 28–30 °C and 220 rpm until OD reached. 600 The result was 10 (48 h). The cells were centrifuged at 3500 g for 5 min at room temperature, collected, and resuspended in BMMY medium to adjust the OD value. 600 The culture medium was 10, and the culture was placed on a shaker at 28℃ and 220rpm for 3 days. Anhydrous methanol was added to the culture medium every 24 hours until the final concentration was 1.0%. Bacterial culture samples were collected at 48h and 72h after methanol induction. The sample volume was 1mL and placed in a 1.5mL EP tube. The tube was centrifuged at 12000g for 5min at 4℃ and the expression supernatant was collected. The samples to be tested were stored at -80℃ for later use.

[0045] The results of SDS-PAGE analysis of the supernatant are as follows: Figure 2 As shown in A, the results indicate that collagen sequences can be secreted into the culture medium supernatant, and the bands are relatively uniform.

[0046] 4. Fermentation preparation of recombinant collagen The Pichia pastoris-COL4-4 ​​genetically engineered yeast obtained in step 3 was used for high-density fermentation to express recombinant collagen on a large scale. A fed-batch culture method was used, and the culture temperature was 28℃. The steps are as follows: (1) Pichia pastoris genetic engineering was inoculated into 1L shake flasks containing 100mL seed culture medium YPD and cultured at 220rpm and 28℃ for 22-24h until the OD600 reached 8-12; (2) Use a 5L fermenter, fill with 2L of fermentation medium, adjust the rotation speed to 600rpm, the aeration rate to 3L / min, and the temperature to 28℃ before inoculation. Adjust the pH with the alkaline solution prepared with concentrated ammonia water and set the pH to 5. Inoculate 100mL of the prepared seed liquid into the tank (inoculate with a flame ring), click the dissolved oxygen electrode to calibrate to 100%, and start fermentation after calibration. (3) When the dissolved oxygen level drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain it at 30%. Wait for the glycerol to be depleted, the dissolved oxygen to rebound, and the dissolved oxygen to be greater than 60% (OD600 value of about 100). Then, induce with methanol, setting the feeding rate to 6 mL / h. After two hours, increase it to 10 mL / h, and finally to 16 mL / h. During the induction period, maintain the temperature at 28℃, adjust the speed to 800 rpm, control the dissolved oxygen at 20%, and maintain the pH at 5 by feeding with ammonia. After about 96 hours of induction, the protein can be removed from the tank if SDS-PAGE shows that the protein has begun to degrade or if UV measurement shows that the protein concentration has not increased significantly.

[0047] Collect the fermentation supernatant and perform SDS-PAGE electrophoresis for analysis. The results are as follows: Figure 2 As shown in B, under high-density fermentation conditions, after 72 hours of induction, the collagen COL4-4 ​​bands were relatively simple, and the proportion of the main bands in the optical density analysis all exceeded 80%.

[0048] 5. Purification of recombinant collagen Collect the fermentation supernatant prepared in step 1, and dilute the fermentation broth with purified water until the conductivity reaches 5.0 mS / cm. Add 1 M citric acid solution to adjust the pH of the fermentation broth to 4.00. Equilibrate the cation exchange medium (chromatographic packing material: Changzhou Tiandi Renhe SP Big Beads, loaded in Jiaxing Dongfulong Qianchun Biotechnology QCXKC-16 / 20) with buffer A until the absorbance and conductivity at 220 nm wavelength are stable. Start loading the sample at a flow rate of 5 mL / min and a loading volume of 0.3 L / time. Detect the absorbance at 220 nm wavelength. When the absorbance is higher than 100 mAu, collect the flow-through fermentation supernatant.

[0049] After sample loading, the cationic chromatography medium was equilibrated with buffer A. When the absorbance at 220 nm dropped to 100 mAu, collection of the flow-through fermentation supernatant was stopped. After equilibration with buffer A until the UV absorbance and conductivity stabilized, the proportion of buffer B was adjusted to 10% for washing, and then adjusted to 50% for elution of the target protein. Protein solution collection began when the 220 nm UV absorbance was above 50 mAu. The protein composition of the washing and elution buffers was analyzed. The target protein solution was concentrated by ultrafiltration until the conductivity was below 0.4 mS / cm. The ultrafiltration concentrate was then filtered through a 0.22 μm sterile filter and freeze-dried to obtain collagen solid (lyophilized powder, the actual form depends on the specific morphology; a loose, porous structure is usually referred to as a sponge).

[0050] In this invention, buffer A comprises citric acid and sodium citrate, and is a 20 mM citrate buffer with pH 4.00. Buffer B comprises citric acid, sodium citrate, and NaCl, with a citrate concentration of 20 mM, a NaCl concentration of 1 M, and a pH of 4.00.

[0051] Example 2 To screen for the number of repetitions, experiments with different numbers of repetitions were conducted under the same conditions as in Example 1. Recombinant collagen single chains with 2, 3, 5, 6, 7, and 10 repetitions were constructed and named COL4-2, COL4-3, COL4-5, COL4-6, COL4-7, and COL4-10, respectively. Expression, purification, and performance testing were performed (other aspects were the same as in Example 1, and the detection methods were the same as in Test Example 1). The results are shown in Table 1.

[0052] Table 1 Performance Table for Different Number of Repetitions

[0053] Table 1 shows that the number of repetitions significantly affects the expression stability and biological activity of recombinant collagen. With two repetitions (COL4-2), the molecular weight was small, resulting in poor expression stability; after 96 hours of fermentation, the main band proportion was only 65%, and thermal stability was poor, with low cell adhesion and proliferation rates. Stability and activity improved with three repetitions. With four repetitions (COL4-4), the overall performance was optimal: the main band proportion reached 93%, it remained stable after heating at 60℃ for 4 hours, the cell adhesion rate reached 107%, and the cell proliferation rate reached 140%, all the highest values ​​among all repetitions. With five and six repetitions, stability remained good, but adhesion and proliferation rates decreased slightly. With seven repetitions, the main band proportion decreased to 85%, and activity further decreased. With ten repetitions (COL4-10), the excessively large molecular weight led to increased expression pressure or protein misfolding, causing the main band proportion to decrease to 58%, with significant degradation and activity reaching its lowest point. Therefore, the preferred number of recombinant collagen single-chain repeats in this invention is 3 to 7 times, with 4 times being the optimal number.

[0054] Example 3 Filtering different combinations of segments: COL4-A: Amino acids 918-938 are repeated 4 times only; COL4-B: Amino acids 1115-1147 are repeated 4 times only; COL4-C: Amino acids 1032-1055 are repeated 4 times only; COL4-Rev: 1032-1055 + 1115-1147 + 918-938, repeated 4 times; COL4-Control (2R2-12, amino acid sequence as shown in SEQ ID NO.5, nucleotide encoding this amino acid as shown in SEQ ID NO.6): SEQ ID NO.5: GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKK REAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKREAGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGL SEQ ID NO.6: Expression, purification, and performance testing were performed (other aspects were the same as in Example 1, and the testing methods were the same as in Test Example 1), and the results are shown in Table 2.

[0055] Table 2. Effects of different segments

[0056] Table 2 shows that different fragment combinations significantly affect the performance of recombinant collagen. Samples containing only a single functional fragment (COL4-A, COL4-B, COL4-C) exhibited some expression stability (main band percentage 76%–81%) and thermal stability, but their cellular biological activity was low, with relative cell adhesion rates of only 65%–70% and relative cell proliferation rates of only 70%–75%. This indicates that single short peptide fragments derived from human type IV collagen lack synergistic effects and are insufficient to mimic the complex functions of natural proteins.

[0057] Combining the three fragments selected in this invention but reversing their order (COL4-Rev, i.e., 1032-1055→1115-1147→918-938) resulted in improved cell adhesion rate (85%) and proliferation rate (95%) compared to the single fragments, but slight degradation occurred (the main band accounted for 88%, and slight degradation occurred at 60℃ / 4h), and the structural stability was not as good as the preferred order of this invention.

[0058] In comparison, the preferred sequence of COL4-4 ​​(918-938→1115-1147→1032-1055, repeated 4 times) of this invention exhibits the best overall performance: the proportion of main bands is as high as 93% after 72 hours of fermentation, with good thermal stability (stable at 60℃ / 4h), a relative cell adhesion rate of 107%, and a relative cell proliferation rate of 140%, all of which are the highest among all samples.

[0059] Furthermore, the fermentation stability of COL4-4 ​​is far superior to that of the COL4-control containing linker peptides (93% vs 55% of the main band), proving that the direct repeatable design without linker peptides in this invention fundamentally avoids the protein degradation problem caused by uncontrollable endogenous enzyme cleavage.

[0060] Example 4 Collagen hemostatic dressings were prepared using the recombinant collagen obtained in Example 1. Preparation method: 1. Dissolve the COL4-4 ​​lyophilized sponge prepared in Example 1 in water for injection to prepare a 2.5% (w / v) collagen solution. Stir magnetically or at low speed (4°C) to ensure complete dissolution and avoid generating bubbles.

[0061] 2. Weigh out the chitosan, dissolve it in 1% acetic acid solution to prepare a 3.0% chitosan solution, and filter to remove insoluble matter.

[0062] 3. Mix the collagen solution and chitosan solution at a volume ratio of 8:2 and stir slowly until homogeneous; add glycerol or sorbitol to a final concentration of 0.5% and stir until homogeneous; add glutaraldehyde (final concentration 0.01%) and stir for 30 minutes to allow it to react fully, and the product is ready.

[0063] Example 5 Collagen compound gel dressing was prepared using the recombinant collagen obtained in Example 1: It is divided into two parts, A and B. Part A is a sterile freeze-dried sponge containing type IV recombinant collagen from Example 1, with a content of 4 mg. Part B consists of the following components by mass percentage: sodium hyaluronate 0.3%, carbomer 1%, propylene glycol 2.0%, glycerin 2.5%, methylparaben 0.05%, propylparaben 0.05%, and the remainder is purified water. When using, mix parts A and B thoroughly.

[0064] The preparation method is as follows: (1) Accurately weigh carbomer, sodium hyaluronate, glycerin, propylene glycol, methylparaben and propylparaben, stir for 1 hour, add purified water and dissolve completely.

[0065] (2) Accurately weigh the freeze-dried type IV recombinant collagen.

[0066] (3) Filling under aseptic conditions, sealing and packaging, and irradiation sterilization result in type IV recombinant collagen compound gel dressing product.

[0067] Instructions for use: Bottles A and B can be aseptically and quickly mixed before use.

[0068] Example 6 Collagen liquid dressing was prepared using the recombinant collagen obtained in Example 1: Type IV recombinant collagen 5mg / mL, the remaining components by mass percentage include: sodium hyaluronate 0.05%, glycerol 10%, carbomer 2%, and the balance being purified water.

[0069] The preparation method is as follows: (1) Weigh carbomer, sodium hyaluronate and glycerin accurately, stir for 1 hour to dissolve completely.

[0070] (2) Accurately weigh the freeze-dried type IV recombinant collagen, add purified water, and dissolve it completely.

[0071] (3) After mixing, fill under aseptic conditions, seal and package, and sterilize by irradiation to obtain the type IV recombinant collagen liquid dressing product.

[0072] How to use: After cleansing, apply evenly to the face and gently massage or pat until fully absorbed.

[0073] Comparative Example 1 Collagen hemostatic dressings were prepared using collagen obtained from COL4-control (same as Example 4).

[0074] Comparative Example 2 Collagen compound gel dressings were prepared using collagen obtained from COL4-control (same as Example 5).

[0075] Comparative Example 3 The gene encoding SEQ ID NO.3 (SEQ ID NO.4) was cloned into the E. coli expression vector (pET-28a), transformed into BL21(DE3), and induced to express intracellularly. The expression was carried out according to the following steps: cell disruption → centrifugation to collect inclusion bodies or supernatant → Ni-NTA affinity chromatography (if His tag is present) → TEV digestion → purification again to remove the tag and enzyme (method of Example 1, CN114940712A).

[0076] Purification: Purification was performed according to the cation exchange chromatography method in step 5 of Example 1: The supernatant was adjusted to pH 4.00 and conductivity to 5.0 mS / cm, and then loaded onto an SP Big Beads cation exchange column equilibrated with buffer A. After equilibration with buffer A, the target protein was eluted sequentially with a solution containing 10% buffer B and a solution containing 50% buffer B. The eluent was collected, concentrated by ultrafiltration, sterilized by filtration, and then freeze-dried to obtain collagen sponge.

[0077] Test Example 1 The recombinant collagen lyophilized collagen sponge prepared in Example 1 was used to test its properties: 1. Thermal stability Recombinant protein lyophilized sponge (COL4-4) was prepared into a 10 mg / mL solution with ultrapure water, filtered through a 0.22 μm filter membrane, and then aliquoted into sterile centrifuge tubes. The tubes were heated at 60 °C for 1 h, 2 h, 3 h, and 4 h, and samples were taken for SDS-PAGE analysis.

[0078] The stability results of COL4-4 ​​are as follows: Figure 3 As shown, the results indicate that the treated COL4-4 ​​still maintains structural and purity stability, with no significant difference from day 0; the results also show that the protein structure was not destroyed at 60℃, and no serious degradation or diffusion occurred, proving that COL4-4 ​​has high thermal stability.

[0079] 2. Acid and alkali tolerance stability test The stability of COL4-4 ​​under different pH conditions was tested, and the results are as follows: Figure 4As shown in A and B, the results indicate that COL4-4 ​​has good pH suitability, maintaining strong stability at pH 6-8, and has excellent clinical pH suitability.

[0080] 3. Cell adhesion and proliferation activity NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GMN6; culture and passage methods were performed according to the cell instructions) were cultured normally. Recombinant collagen lyophilized sponge COL4-4 ​​was added to each well of a 96-well cell culture plate at concentrations of 1, 2, and 4 mg / mL, along with 100 μL of each protein solution and a blank PBS control. The plate was incubated at room temperature for 60 min. Then, 10 μL of the solution was added to each well. 5 3T3 cells in good culture condition were incubated at 37°C and 5% CO2 for 60 min; the cells were washed 4 times with PBS.

[0081] The determination of recombinant collagen adhesion and proliferation was carried out in accordance with the YY1849-2022 industry standard for recombinant collagen.

[0082] Higher adhesion activity means that collagen can help cells adhere to the cell wall or extracellular matrix more quickly, thus better constructing the cell growth microenvironment and maximizing the effects of collagen. Results are as follows: Figure 5 As shown, the results indicate that the concentration of COL4-4 ​​at 2 mg / ml was significantly higher than that in the control group.

[0083] Higher proliferation activity indicates that collagen, while providing a microenvironment, can also stimulate cell regeneration, resulting in better proliferation and thus stronger collagen activity. The results are as follows: Figure 6 As shown, the proliferation activity of COL4-4 ​​at 2 mg / ml and 4 mg / ml was significantly higher than that of the control group.

[0084] 4. Safety The testing should be conducted according to GB / T16886.5-2017, and the steps are as follows: The potential cytotoxic effects of recombinant collagen COL4-4 ​​were tested using in vitro cultured mammalian L-929 cells. Recombinant collagen (1 mg / mL, 2 mg / mL, 4 mg / mL) and control samples were placed in MEM medium containing 10% fetal bovine serum and incubated at 37°C for 24 hours. After extraction, 96-well plates (103 wells) cultured for 24 hours were... 4 Remove the culture medium from each cell (cells / well), replace it with the appropriate extraction solution, and incubate in a cell culture incubator (37℃, 5% CO2, >90% humidity) for 24 hours.

[0085] After culture, cell morphology and cell lysis were observed under a microscope, and the cytotoxicity value of the test sample was determined using the CCK8 assay. Results are as follows: Figure 7As shown, the results indicate that the blank control group and cells maintained normal morphology throughout the experiment and showed no cytotoxic reaction. After incubation at a concentration of 4 mg / mL for 24 hours, the cell morphology remained basically intact, and the cell viability was 90%. This shows that recombinant collagen COL4-4 ​​did not have any cytotoxic effect under the CCK8 cytotoxicity assay conditions.

[0086] 5. Endotoxin and immunogenicity tests (1) The determination shall be performed according to the gel method or dynamic turbidity method in Section 1143 "Bacterial Endotoxin Test Method" of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0087] (2) In vitro immune stimulation test Mouse macrophages RAW264.7 were seeded in 24-well plates (density 2 × 10⁻⁶). 5 Cells / well were cultured overnight. Different concentrations of test solution (e.g., 0.1, 1, 10 μg / mL) were added, and lipopolysaccharide (LPS, 1 μg / mL) was set as a positive control. Cells were cultured for 24 hours. Cell supernatant was collected, and the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was detected according to the ELISA kit instructions.

[0088] Endotoxin and immunogenicity tests were performed on the collagen obtained from COL4-control, Comparative Example 3, and Example 1. The results are shown in Table 3.

[0089] Table 3. Endotoxin content and immune stimulation results of different collagen samples

[0090] Note: Compared with the negative control, p<0.05, p>0.05 (no significant difference). TNF-α and IL-6 release levels were measured after RAW264.7 macrophages were co-incubated with a 100 μg / mL sample.

[0091] As shown in Table 3, both COL4-4 ​​and COL4-control in Example 1 were expressed by Pichia pastoris, and their endotoxin content was less than 0.05 EU / mg, meeting the stringent requirements for low endotoxin in medical devices and injectables. Although Comparative Example 3 (E. coli product) underwent the same cation exchange purification, its endotoxin content was still as high as 4.8 EU / mg. This demonstrates that despite multi-step purification, the residual amount was still much higher than that of the yeast expression system due to the tight binding of endotoxin to proteins or the formation of aggregates. After stimulation with COL4-4 ​​in Example 1, the levels of TNF-α and IL-6 released by macrophages were not significantly different from the negative control (p>0.05), demonstrating extremely low immunogenicity risk and excellent biocompatibility. The E. coli product in Comparative Example 3 induced significant cytokine release, which is related to its high endotoxin content and potential impurities. Although the COL4-control had low endotoxin, its induced cytokine release level was significantly higher than that of Example 1 and the negative control (p<0.05). This indicates that even if the endotoxin is qualified, non-natural linker peptides (such as KKREA) or fragments thereof that may remain in the COL4-control due to incomplete enzymatic cleavage may still be recognized by immune cells, triggering low-level immune stimulation and increasing the safety risk of the product.

[0092] Test Example 2 An evaluation experiment was conducted on the healing of diabetic wounds using the products of Example 5 and Comparative Example 2: Fifty healthy SPF-grade SD rats, weighing between 230-250g, were used. They were acclimatized for one week before the experiment, housed at a temperature of 20±3℃ and a relative humidity of 55±10%, with free access to food and water. Before modeling, each rat was fasted for 12 hours and injected with 1% STZ solution (prepared with citrate buffer) at a dose of 50mg / kg. Thirty minutes after injection, food and water intake were no longer restricted. Blood glucose was measured from the tail vein 72 hours after injection. A blood glucose level ≥16.7mM indicated successful modeling; rats that did not successfully model were excluded from the experiment. Rats with successful modeling were divided into three groups of 15 each.

[0093] A 2cm diameter circle was cut on the back of a rat to obtain a skin lesion. The collagen compound gel dressing obtained in Example 5 and Comparative Example 2 was applied to the skin lesion twice a day for four weeks. The control group was treated with a phosphate buffer solution with pH=7.0. The diameter of the skin lesion was measured to confirm the wound healing effect. The results are as follows Figure 8 As shown, by Figure 8 It can be seen that the collagen compound gel dressing prepared in Example 5 of the present invention has a good effect on promoting wound healing, and the wound healing rate reaches 98% after 28 days. Compared with the control group, the gel in Comparative Example 2 is effective compared with the blank control, but the effect is worse than that in Example 5.

[0094] Test Example 3 The hemostatic effect of the products from Example 4 and Comparative Example 1: Thirty 5-week-old Kunming mice, weighing 25±2g, were randomly divided into three groups, half male and half female, with 10 mice in each group. Each mouse was anesthetized by injecting 3% sodium pentobarbital at a dose of 30mg / kg. The mice were then fixed, and the tails were cut off 1cm from the end with surgical scissors. Any bleeding was treated immediately with hemostatic materials. The blank group used physiological saline, while the groups in Example 4 and Comparative Example 1 used the hemostatic dressings from Example 4 and Comparative Example 1, respectively. The positive control group was treated with Yunnan Baiyao powder. Timing was started from the tail cut, and bleeding was considered stopped after 20 minutes. The results are shown in Table 4.

[0095] Table 4. Effects of different products on hemostasis time in mice

[0096] As shown in Table 4, in the mouse tail amputation hemostasis model, the collagen hemostatic dressing prepared in Example 4 of this invention performed excellently, with an average hemostasis time of 120 seconds, significantly better than the positive control group and the blank control group. In contrast, the dressing prepared using COL4-control (containing linker peptides) in Comparative Example 1 had a hemostasis time of 155 seconds, which was better than the blank control, but significantly inferior to Example 4. This result is consistent with the cell activity data (Table 2) and safety data (Table 3) in Test Example 1, demonstrating that COL4-4 ​​of this invention has significant advantages in the preparation of hemostatic materials due to its high stability, high activity, and high safety.

[0097] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A type IV recombinant collagen, characterized in that, The recombinant collagen comprises a recombinant collagen single chain; wherein the recombinant collagen single chain is obtained by repeating the collagen monomer fragment as shown in SEQ ID NO.2 2 to 10 times.

2. The type IV recombinant collagen according to claim 1, characterized in that, The recombinant collagen monomer fragments are obtained by cutting and splicing the amino acid sequence of human type IV collagen; wherein the amino acid sequence of human type IV collagen is shown in SEQ ID NO.

1.

3. The type IV recombinant collagen according to claim 2, characterized in that, The recombinant collagen monomer fragment is obtained by sequentially combining amino acids at positions 918-938, 1115-1147, and 1032-1055 of human type IV collagen; wherein the amino acid sequence of human type IV collagen is shown in SEQ ID NO.

1.

4. The type IV recombinant collagen according to claim 3, characterized in that, The nucleotide sequence encoding the recombinant collagen is shown in SEQ ID NO.

4.

5. A method for preparing type IV recombinant collagen, characterized in that, The method includes: Step 1: Design collagen sequences The amino acid sequences of human type IV collagen, as shown in SEQ ID NO. 1, are sequentially combined at positions 918-938, 1115-1147, and 1032-1055 to obtain recombinant collagen monomer fragments; the recombinant collagen single chains are obtained by repeating the collagen monomer fragment shown in SEQ ID NO. 2 2 to 10 times. Step 2, constructing the carrier Based on the complete nucleotide sequence of the recombinant collagen single strand, it is recombined into a plasmid to obtain a recombinant plasmid; Step 3: Transformation and Cultivation The recombinant plasmid was transferred into a eukaryotic host cell, and engineered strains were screened. The engineered strains were then fermented and cultured to induce the expression of the target protein under appropriate conditions. Step 4: Harvesting and Purification The fermentation broth was collected, the expressed target protein was harvested, and the protein product was purified to obtain high-purity target collagen.

6. The method for preparing type IV recombinant collagen according to claim 5, characterized in that, In step two, the plasmids used are prokaryotic expression plasmids, specifically pET series vectors, pGEX series vectors, pBAD series vectors, or pUC series vectors.

7. The method for preparing type IV recombinant collagen according to claim 6, characterized in that, In step two, the plasmids used are eukaryotic expression plasmids, specifically pcDNA series vectors, pCMV series vectors, pIRES series vectors, pEGFP series vectors, pYES series vectors, or pPIC series vectors.

8. The method for preparing type IV recombinant collagen according to claim 5, characterized in that, In step three, the host cell is a yeast cell or a mammalian cell.

9. The use of type IV recombinant collagen prepared by the method according to any one of claims 5-8 in the preparation of collagen-containing products, characterized in that, The collagen products mentioned are cosmetics, medical devices, or pharmaceuticals.

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