Stable expression of V-type recombinant collagen and preparation method and application thereof

Abstract

The application discloses stable V-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 recombinant collagen protein prepared by the application has excellent stability, can still maintain structural and purity stability after being heated at 60 DEG C for 4 hours, and has good acid-base (pH 6-8) resistance.

Description

Technical Field

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

[0002] Collagen is the most abundant structural protein in mammals. While V-type collagen accounts for only 2%-5% of total collagen in the skin, it plays a crucial role as the "molecular commander" regulating fiber assembly. Through co-assembly with type I and type III collagen, it utilizes steric hindrance to limit excessive fiber thickening, ensuring the formation of a fine and uniform network structure, thereby maintaining skin firmness and elasticity. Studies have shown that V-type collagen deficiency leads to disordered fiber arrangement and abnormally increased diameter, directly causing skin sagging and wrinkles. Therefore, developing highly active recombinant humanized V-type collagen has become a core requirement in the anti-aging field.

[0003] However, existing strategies for preparing recombinant type V collagen struggle to balance expression stability and bioactivity. Chinese patent CN116478274A employs tandem repeats of the natural continuous amino acid sequence of human type V collagen to preserve its natural conformation. While the product exhibits excellent bioactivity and significantly upregulates collagen expression, it suffers from serious drawbacks in industrial eukaryotic expression systems such as Pichia pastoris: the protease recognition sites implicit in the continuous sequence are amplified after repetition, leading to the target protein being highly susceptible to non-specific degradation. In actual production, the yield of full-length protein is often low, accompanied by numerous degradation bands, which not only increases purification difficulty and cost but also severely affects product uniformity and safety, making large-scale stable production difficult.

[0004] To improve stability, Chinese patent CN118388632A uses ultrashort peptides less than 16 amino acids in length for high-frequency repetition. While this design effectively avoids enzyme cleavage sites and improves expression stability, the recombinant protein is too short to fold into the triple helix structure and key functional domains characteristic of V-type collagen. This results in a significant reduction in its biological activity, failing to effectively mimic the complex regulatory functions of natural V-type collagen and thus failing to meet the needs of high-end anti-aging and regenerative medicine.

[0005] Therefore, there is an urgent need to develop a novel recombinant V-type collagen that can overcome the limitations of existing designs based on long continuous sequences, such as poor stability and easy degradation during microbial expression, and avoid the drawbacks of functional incompleteness of ultrashort peptide sequences. This would enable high-quality, low-cost, large-scale preparation to meet the urgent needs of the biopharmaceutical and high-end cosmetic industries. Summary of the Invention

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

[0007] This invention primarily addresses the issues of recombinant collagen being easily degraded, exhibiting increased immunogenicity, and low biological activity during expression in Pichia pastoris. By selecting the full-length or partial genes of natural human collagen and optimizing their combination, this invention designs a collagen amino acid sequence with high stability and biological activity. Theoretically, this amino acid sequence is completely consistent with the corresponding region sequence of natural human collagen, exhibiting low immunogenicity while maintaining the typical biological functions of human collagen.

[0008] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a recombinant V-type collagen protein with stable expression is provided, wherein 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 2 to 10 times. As an example, it is preferable to repeat 3 to 7 collagen monomer fragments; more preferably, it is repeated 4 to 5 times. Although the monomer fragments designed in this invention have undergone sequence rearrangement, the three selected fragments are all derived from the key functional domains of human V-type collagen, and the total length is moderate, and a stable higher-order structure can be formed after repeating 4 to 5 times.

[0009] Furthermore, the recombinant collagen single chain is obtained by cutting and splicing the amino acid sequence of human type V collagen; wherein the amino acid sequence of human type V collagen is shown in SEQ ID NO. 1.

[0010] SEQ ID NO. 1: The amino acid sequence of this invention is entirely derived from a combination of the natural human V-type collagen sequence, without introducing any non-human exogenous amino acid residues. Theoretically, its sequence is completely consistent with the corresponding region in the human body, thus exhibiting extremely low immunogenicity and avoiding the risk of viruses carried by animal-derived collagen and allergic reactions caused by foreign proteins.

[0011] Furthermore, the recombinant collagen monomer fragment is composed of amino acids at positions 1360-1380, 982-1011, and 1405-1434 of human type V collagen. This invention innovatively selects three discontinuous fragments at positions 1360-1380, 982-1011, and 1405-1434 of human type V collagen for artificial splicing, constructing a unique monomer fragment (amino acid sequence as shown in SEQ ID NO. 2).

[0012] SEQ ID NO. 2: GPPGDKGDDGEPGQTGSPGPTGETGFQGKTGPPGPPGVVGPQGPTGETGPMGEKGAKGEAGLEGPPGKTGPIGPQGAPGKP; This "cross-region splicing" strategy effectively disrupts specific protease recognition sites and unstable local secondary structures hidden in natural continuous sequences.

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

[0014] According to a second aspect of the present invention, a method for preparing a stable V-type recombinant collagen is provided, the method comprising: Step 1: Design collagen sequences The amino acid sequences of human type V collagen, as shown in SEQ ID NO. 1, are sequentially combined at positions 1360-1380, 982-1011, and 1405-1434 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 the host cell, and the engineered strain was screened. The engineered strain was then fermented and cultured, and the expression of the target protein was induced under appropriate conditions. Step 4: Harvesting and Purification The fermentation broth was collected, and the expressed protein product was harvested. High-purity target collagen was obtained by efficient purification using cation exchange or a combination of cation exchange and hydrophobic chromatography.

[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 are pET-28a, pET-32a, pGEX-4T-1, pBAD / Myc-His, or 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 an example, preferred plasmids include pcDNA3.1, pCMV-Tag, pCMV-Sport, pIRES2-EGFP, pEGFP-C1, pYES2, pPICZ, or pPIC3.5.

[0017] Furthermore, in step three, the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell. For example, the prokaryotic cell is *Escherichia coli* or *Bacillus subtilis*; the yeast cell is *Pichia pastoris* or *Saccharomyces cerevisiae*; and the mammalian cell is HEK293 cell or CHO cell.

[0018] The third aspect of the present invention provides the application of a stable V-type recombinant collagen in the preparation of collagen-containing products.

[0019] Furthermore, the collagen product is a cosmetic or medical device.

[0020] Furthermore, the collagen product is a drug.

[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] The present invention has the following advantages: (1) After heating at 60°C for 4 hours, SDS-PAGE analysis showed no significant changes in the structure and purity of the protein of this invention, indicating no degradation or aggregation. This characteristic not only reduces cold chain costs during production, transportation, and storage but also enables it to withstand common sterilization or drying processes in medical device manufacturing. It exhibits extremely strong stability within the physiological and formulation-common pH range of 6-8. Due to the extremely low impurity content in the fermentation broth, a product with a purity >93% (HPLC detection) can be obtained through a single cation exchange chromatography step, significantly simplifying downstream purification processes, improving yield, and making it suitable for large-scale industrial preparation.

[0026] (2) The recombinant collagen prepared in this invention has excellent stability during Pichia pastoris expression. After fermentation in 5L BSM medium, the bands are single and there are no obvious degradation bands.

[0027] (3) The recombinant collagen prepared by this invention has high adhesion and proliferation activity. Compared with the control, after 24 hours of incubation at 4 mg / ml COL5-5, the cell morphology is basically intact, the cell viability is above 92%, there is no cytotoxicity, and the biocompatibility is high.

[0028] (4) The amino acid sequence of this invention is entirely derived from the cutting and splicing of natural fragments of human type V collagen, without introducing any exogenous amino acid residues or non-natural linker peptides. Therefore, it fundamentally eliminates the risk of animal-derived collagen carrying viruses and greatly reduces the risk of immunogenicity and allergic reactions caused by foreign proteins, providing a very high level of safety for high-end medical aesthetics, regenerative medicine and clinical treatment. Attached Figure Description

[0029] 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.

[0030] 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.

[0031] Figure 1 This is a schematic diagram of the recombinant plasmid provided in Embodiment 1 of the present invention; Figure 2 The image shows the recombinant collagen expression electrophoresis diagram provided in Example 1 of the present invention. In this image, A is the SDS-PAGE detection result of the supernatant of recombinant collagen expression in Pichia pastoris genetically engineered bacteria; B is the SDS-PAGE detection result of the supernatant from 5L large-scale fermentation, with the left side diluted 20 times and the right side diluted 10 times before loading. Figure 3 The results of SDS-PAGE detection during the purification process of recombinant collagen provided in Example 1 of this invention; Figure 4 The results are HPLC analysis of the purified and lyophilized recombinant collagen provided in Example 1 of this invention. Figure 5 The results show the thermal stability of the recombinant collagen COL5-5 provided in Test Example 1 of this invention; Figure 6 The results of the acid-base tolerance test of recombinant collagen COL5-5 provided in Test Example 1 of this invention; Figure 7 The results of the recombinant collagen cell adhesion activity test provided in Test Example 1 of this invention; Figure 8The results of the recombinant collagen cell proliferation activity detection provided in Test Example 1 of this invention; Figure 9 The results of the recombinant collagen cytotoxicity test provided in Test Example 1 of this invention; Figure 10 The image shows the evaluation results of diabetic wound healing provided in Test Example 2 of this invention. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Example 1 This embodiment provides a stable V-type recombinant collagen expression: 1. Design collagen sequences Based on the sequence of human type V collagen (COL5A1, amino acid sequence as shown in SEQ ID NO.1), recombinant collagen was designed as follows: The amino acids at positions 1360-1380, 982-1011, and 1405-1434 were combined sequentially to obtain recombinant collagen monomer fragment 1, the amino acid sequence of which is shown in SEQ ID NO.2. SEQ ID NO.2 was repeated 5 times to obtain a recombinant collagen single chain, named 5-5, the amino acid sequence of which is shown in SEQ ID NO.3.

[0036] SEQ ID NO.3: GPPGDKGDDGEPGQTGSPGPTGETGFQGKTGPPGPPGVVGPQGPTGETGPMGEKGAKGEAGLEGPPGKTGPIGPQGAPGKPGPPGDKGDDGEPGQTGSPGP TGETGFQGKTGPPGPPGVVGPQGPTGETGPMGEKGAKGEAGLEGPPGKTGPIGPQGAPGKPGPPGDKGDDGEPGQTGSPGPTGETGFQGKTGPPGPPGVVGP QGPTGETGPMGEKGAKGEAGLEGPPGKTGPIGPQGAPGKPGPPGDKGDDGEPGQTGSPGPTGETGFQGKTGPPGPPGVVGPQGPTGETGPMGEKGAKGEAG LEGPPGKTGPIGPQGAPGKPGPPGDKGDDGEPGQTGSPGPTGETGFQGKTGPPGPPGVVGPQGPTGETGPMGEKGAKGEAGLEGPPGKTGPIGPQGAPGKP; 2. Construction of recombinant plasmids and recombinant bacteria Take the amino acid sequence SEQ ID NO.3 obtained in step 1 and entrust Nanjing Genscript Biotech Co., Ltd. to complete the corresponding nucleotide sequence (corresponding to nucleotide sequence SEQ ID NO.4 respectively). SEQ ID NO.4: The plasmid was then inserted into the α-factor reading frame of the empty pPICzα vector (purchased from Thermo Fisher Scientific) to obtain the recombinant plasmid, named pPICzα-COL5-5 (also known as COL5-α1_pPICZalphaA), with the amino acid sequence SEQ ID NO. 3 and the nucleotide sequence SEQ ID NO. 4. A schematic diagram of the plasmid is shown below. Figure 1 As shown.

[0037] 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-COL5-5.

[0038] 3. Induced expression and identification of recombinant collagen The Pichia pastoris genetically engineered bacteria obtained in step 2 were inoculated into 50 mL centrifuge tubes containing 5 mL of BMGY medium and cultured at 28-30℃ and 220 rpm until the OD600 reached 10 (48 h). The cells were centrifuged at 3500 g for 5 min at room temperature, and the cells were collected. The cells were resuspended in BMGY medium to approximately OD600 of 10 and placed on a shaker at 28℃ and 220 rpm for 3 days of continued growth. Anhydrous methanol was added to the medium every 24 h until the final concentration reached 1.0%. Bacterial samples were collected at 48 h and 72 h after methanol induction, with a sample volume of 1 mL, placed in a 1.5 mL EP tube, and centrifuged at 12000 g for 5 min at 4℃. The supernatant was collected, and the samples to be tested were stored at -80℃ for later use.

[0039] The results of SDS-PAGE analysis of the supernatant are as follows: Figure 2 As shown in Figure A, the results indicate that the designed V-type collagen sequence can be secreted into the culture medium supernatant, and the bands are clear and uniform.

[0040] 4. Fermentation preparation of recombinant collagen The Pichia pastoris-COL5-5 genetically engineered yeast obtained in Example 1 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°C. The steps are as follows: (1) Pichia pastoris genetic engineering was inoculated into 1L shake flasks containing 100mL of seed culture medium YPD and cultured at 220rpm and 28℃ for 22-24h until OD was reached. 600 Reaching 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 level to be greater than 60% (OD). 600 With a protein concentration of approximately 100, methanol induction was initiated, with a feed rate set at 6 mL / h. After two hours, this was increased to 10 mL / h, and finally to 16 mL / h. During induction, the temperature was maintained at 28℃, the rotation speed was adjusted to 800 rpm, dissolved oxygen was controlled at 20%, and pH was maintained at 5 by adding ammonia. After approximately 96 hours of induction, the protein was discharged from the tank when SDS-PAGE analysis showed that protein degradation had begun or UV measurement showed no significant increase in protein concentration.

[0041] Collect the fermentation supernatant and perform SDS-PAGE electrophoresis for analysis. The results are as follows: Figure 2 As shown in Figure B, under high-density fermentation conditions, after 72 hours of induction, collagen COL5-5 showed almost only the target band, and the proportion of the main band in the optical density analysis exceeded 90%.

[0042] 5. Purification of recombinant collagen Collect the fermentation supernatant prepared in step 1, and dilute the fermentation broth with purified water until the conductivity is 5.0 mS / cm. Add 1 M citric acid solution to adjust the pH of the fermentation broth to 3.50. 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. Measure the absorbance at 220 nm wavelength. When the absorbance is higher than 100 mAu, collect the flow-through fermentation supernatant.

[0043] After sample loading, the cation exchange 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 5% for washing, and then adjusted to 50% for elution of the target protein. Protein solution collection began when the 220 nm UV absorbance exceeded 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 the final product.

[0044] The SDS-PAGE electrophoresis results of the COL5-5 purification process are as follows: Figure 3 As shown, the purity was determined by HPLC. Figure 4 As shown, the purity is >93%. In this invention, buffer A comprises citric acid and sodium citrate, and is a 20 mM citrate buffer with pH 3.5. 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 3.5.

[0045] 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, 4, 6, 7, and 10 repetitions were constructed and named COL5-2, COL5-3, COL5-4, COL5-6, COL5-7, and COL5-10, 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.

[0046] Table 1 Performance Table for Different Number of Repetitions

[0047] Table 1 shows that the proportion of the main band initially increases and then decreases with increasing repetition count. The expression stability is optimal at 5 repetitions (93% main band proportion); 4 repetitions (92%) are similar; stability begins to decline after 6 repetitions; and at 10 repetitions, the main band proportion is only 65%, indicating significant degradation. This suggests that moderate repetition (around 5 times) is beneficial for stable expression, while excessive repetitions amplify underlying unstable factors or protease recognition sites in the sequence, leading to non-specific degradation of the target protein. In the stability test after heating at 60℃ for 4 hours, samples with only 4-5 repetitions remained stable, those with 3 and 6 repetitions showed basic stability, while those with 2 and 7 or more repetitions showed varying degrees of degradation, indicating that the number of repetitions also significantly affects thermal stability. From a biological activity perspective, both relative cell adhesion and proliferation rates peaked at 5 replicates (109% and 122%, respectively). Replicas at 4 (107% / 120%) and 6 (105% / 118%) were slightly lower than at 5 replicates, with further decreases at 3 and 7 replicates. The lowest activity was observed at 2 and 10 replicates. Analysis suggests that 5 replicates achieve the optimal balance between functional domain density and spatial conformation. Too few replicates result in insufficient functional domain density, hindering effective mediation of cell-matrix interactions; too many replicates may weaken overall biological function due to interference from degradation products or increased steric hindrance.

[0048] Example 3 Filtering different combinations of segments: COL5-A: Amino acids 1360-1380 repeated 5 times; COL5-B: Amino acids 982-1011 repeated 5 times; COL5-C: Amino acids 1405-1434 repeated 5 times; COL5-Rev: 1405-1434 + 982-1011 + 1360-1380, repeated 5 times; COL5-Nat: 980-1058 of human type V collagen, repeated 5 times; COL5-Short: KPGPRGQRGPTGPRGE (SEQ ID NO.5), repeated 5 times; The corresponding nucleotide after 5 repetitions is SEQ ID NO.6: CTCGAGAAAAGAAAACCAGGACCAAGAGGTCAGCGTGGACCCACAGGACCTAGAGGTGAGAAACCTGGCCCCAGAGGCCAAAGAGGACCTACAGGTCCTCGTGGTGAGAAACCAGGACCTCGTGGTCAGAGGGGTCCAACTGGACCACGAGGTGAGAAGCCAGGTCCACGTGGTCAAAGAGGACCAACTGGACCCAGAGGAGAGAAGCCAGGTCCAAGAGGTCAGCGAGGACCTACCGGACCAAGAGGAGAATAAGCGGCCGC (SEQ ID NO. 6). 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 2.

[0049] Table 2. Effects of different segments

[0050] As shown in Table 2, the COL5-5 of this invention exhibits significantly better expression stability and biological activity than single fragment repeats (COL5-A, B, C), reverse splicing (COL5-Rev), natural continuous long sequence repeats (COL5-Nat), and ultrashort peptides (COL5-Short). The single fragment repeat samples accounted for 76%–81% of the main band. Although their stability was acceptable, their cell adhesion rate (95%–99%) and proliferation rate (105%–109%) were lower than those of COL5-5, indicating that retaining only a single local fragment of type V collagen cannot form a complete functional conformation. The reverse splicing sample COL5-Rev accounted for 86% of the main band and showed slight degradation after heating at 60°C for 4 hours. Although its cell adhesion rate (104%) and proliferation rate (116%) were higher than those of the single fragment, they were still lower than those of COL5-5 of this invention (109% and 122%), indicating that the order of fragment splicing optimizes both stability and activity. The natural continuous long repeat (COL5-Nat) main band accounted for only 55% and degraded significantly after heating at 60°C. Although the purified full-length protein still retained some activity (adhesion rate 106%, proliferation rate 118%), its expression stability was extremely poor, making it difficult to achieve industrial production. The ultrashort peptide COL5-Short showed the highest expression stability (main band accounted for 96%, stable at 60°C), but due to its short sequence (only 16 amino acids), its cell adhesion rate (92%) and proliferation rate (103%) were the lowest among all samples, significantly weaker than those of this invention.

[0051] In summary, this invention achieves optimal biological activity while ensuring high expression stability through the targeted splicing and appropriate repetition (5 times) of three specific fragments.

[0052] Example 4 Collagen hemostatic dressings were prepared using the recombinant collagen obtained in Example 1. Recombinant collagen COL5-5 1.0%~2.0% (w / v), chitosan 0.5%~1.0% (w / v), glycerol or sorbitol 0.5%~2.0% (v / v), cross-linking agent (glutaraldehyde / EDC) 0.01%~0.1% (w / v), and water for injection to bring the total to 100%.

[0053] Preparation method: 1. Dissolve the COL5-5 lyophilized sponge or lyophilized powder 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.

[0054] 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.

[0055] 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.

[0056] 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 sterile lyophilized and contains 4 mg of stable V-type recombinant collagen. Part B consists of the following components by mass percentage: sodium hyaluronate 0.25%, carbomer 1%, propylene glycol 2.5%, glycerin 2%, methylparaben 0.05%, propylparaben 0.05%, and the remainder is purified water. When using, mix parts A and B thoroughly.

[0057] 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.

[0058] (2) Accurately weigh the freeze-dried V-type recombinant collagen.

[0059] (3) Filling under aseptic conditions, sealing and packaging, and irradiation sterilization result in V-type recombinant collagen compound gel dressing product.

[0060] Instructions for use: Bottles A and B can be aseptically and quickly mixed before use.

[0061] Example 6 Collagen liquid dressing was prepared using the recombinant collagen obtained in Example 1: V-type recombinant collagen 5mg / mL, the remaining components by mass percentage include: small molecule sodium hyaluronate 0.05%, glycerol 10%, carbomer 2%, and the balance is purified water.

[0062] The preparation method is as follows: (1) Accurately weigh carbomer, sodium hyaluronate and glycerin, stir for 1 hour to dissolve completely.

[0063] (2) Accurately weigh the freeze-dried V-type recombinant collagen, add purified water, and dissolve it completely.

[0064] (3) After mixing, fill the package under aseptic conditions, seal it, and sterilize it by irradiation to obtain the V-type recombinant collagen liquid dressing product.

[0065] How to use: After cleansing, apply evenly to the face and gently massage or pat until fully absorbed.

[0066] Comparative Example 1 Collagen hemostatic dressings were prepared using collagen obtained from COL5-Nat (same as Example 4).

[0067] Comparative Example 2 Collagen compound gel dressings were prepared using collagen obtained from COL5-Nat (same as Example 5).

[0068] Comparative Example 3 Collagen hemostatic dressings were prepared using collagen obtained from COL5-Short (same as Example 4).

[0069] Comparative Example 4 Collagen compound gel dressings were prepared using collagen obtained from COL5-Short (same as Example 5).

[0070] Test Example 1 The properties of the recombinant collagen freeze-dried collagen sponge obtained in Example 1 were tested: 1. Thermal stability Recombinant protein lyophilized sponge (COL5-5) 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.

[0071] The stability results of COL5-5 are as follows: Figure 5 As shown, the results indicate that the treated COL5-5 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 COL5-5 has extremely high thermal stability.

[0072] 2. Acid and alkali tolerance stability test The stability of COL5-5 under different pH conditions was tested, and the results are as follows: Figure 6 As shown in A and B, the results indicate that COL5-5 has good pH suitability, maintains strong stability at pH 6-8, and has excellent clinical pH suitability.

[0073] 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 COL5-5 was used, and concentrations of 1, 2, and 4 mg / mL were determined. 100 μL of each protein solution and a blank PBS control were added to each well of a 96-well cell culture plate, and the plates were incubated at room temperature for 60 min. Then, 10⁵ well-cultured 3T3 cells were added to each well and incubated at 37°C and 5% CO₂ for 60 min. The cells were washed four times with PBS.

[0074] The determination of recombinant collagen adhesion and proliferation was carried out in accordance with the YY1849-2022 industry standard for recombinant collagen.

[0075] 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 7 As shown, the results indicate that COL5-5 levels at 2 mg / ml and 4 mg / ml were significantly higher than those in the control group.

[0076] 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 8 As shown, the results indicate that the cell proliferation activity of 2 mg / ml COL5-5 was significantly higher than that of the control group.

[0077] 4. Safety The testing should be conducted according to GB / T16886.5-2017, and the steps are as follows: The potential cytotoxicity of recombinant collagen COL5-5 was 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, the cell culture medium in 96-well plates (10⁴ cells / well) was removed, replaced with the corresponding extraction solution, and the plates were incubated for 24 hours in a cell culture incubator (37°C, 5% CO₂, >90% humidity).

[0078] 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 9 As 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 92%. This shows that recombinant collagen COL5-5 had no cytotoxic effect under the CCK8 cytotoxicity assay conditions.

[0079] Test Example 2 An evaluation experiment on the healing of diabetic wounds was conducted using the products of Example 5 and Comparative Examples 2 and 4: Eighty 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 four groups of 15 each.

[0080] A 2cm diameter circle was cut on the back of a rat to obtain a skin lesion. The collagen compound gel dressings obtained in Example 5, Comparative Example 2, and Comparative Example 4 were applied to the skin lesion twice daily for four weeks. The control group was treated with a phosphate buffer solution at pH 7.0. The diameter of the skin lesion was measured to confirm the wound healing effect. The results are as follows Figure 10 As shown, by Figure 10 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 gels of Comparative Examples 2 and 4 are both effective compared with the blank control, and the effect of Comparative Example 2 is better than that of Comparative Example 4; but the effect is worse than that of Example 5.

[0081] Test Example 3 Hemostatic effect of the products from Example 4 and Comparative Examples 1 and 3: Fifty 5-week-old Kunming mice, weighing 25±2g, were randomly divided into 5 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. Group 4, Comparative Example 1, and Comparative Example 3 used the hemostatic dressings of Example 4, Comparative Example 1, and Comparative Example 3, 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 3.

[0082] Table 3. Effects of different products on hemostasis time in mice

[0083] As shown in Table 3, the hemostasis time of Example 4 was the shortest, averaging 130 seconds, significantly better than the positive control group and all comparative groups. Compared with commercially available Yunnan Baiyao, this indicates that the recombinant collagen of the present invention has excellent procoagulant activity. Although the hemostasis time of Comparative Example 1 was faster than the blank control and the positive control, it was slower than that of Example 4 of the present invention. This result indicates that although the long continuous sequence (COL5-Nat) retains the core functional domain of type V collagen and has certain biological activity, its unstable expression and the presence of degradation products during purification affect its practical application effect. The hemostasis time of Comparative Example 3 was the slowest among the three test samples, only slightly better than the positive control group. This indicates that although the ultrashort peptide (COL5-Short) has excellent expression stability (main band accounts for 96%), its short sequence and lack of the functional domain conformation specific to type V collagen (such as cytoin binding domain, heparin binding domain, etc.) result in low biological activity and an inability to effectively promote platelet aggregation and coagulation cascade reactions. Therefore, its in vivo hemostasis effect is significantly weaker than that of the present invention.

[0084] 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 stable V-type recombinant collagen protein, 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 3-6 times.

2. The stable V-type recombinant collagen expressed according to claim 1, characterized in that, The recombinant collagen single chain is obtained by cutting and splicing the amino acid sequence of human type V collagen; wherein the amino acid sequence of human type V collagen is shown in SEQ ID NO.

1.

3. The stable V-type recombinant collagen expressed according to claim 2, characterized in that, The recombinant collagen monomer fragment is obtained by sequentially combining amino acids at positions 1360-1380, 982-1011, and 1405-1434 of human type V collagen.

4. The stable V-type recombinant collagen expressed according to claim 3, characterized in that, The recombinant collagen single chain is obtained by repeating the collagen monomer fragment shown in SEQ ID NO.2 five times, and the nucleotide sequence encoding the collagen is shown in SEQ ID NO.

4.

5. A method for preparing stable V-type recombinant collagen, characterized in that, The method includes: Step 1: Design collagen sequences The amino acid sequences of human type V collagen, as shown in SEQ ID NO. 1, are sequentially combined at positions 1360-1380, 982-1011, and 1405-1434 to obtain recombinant collagen monomer fragments; this process is repeated 3-6 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 the host cell, and the engineered strain was screened. The engineered strain was then fermented and cultured, and the expression of the target protein was induced under appropriate conditions. Step 4: Harvesting and Purification The fermentation broth was collected, and the expressed protein product was harvested. High-purity target collagen was obtained by efficient purification using cation exchange or a combination of cation exchange and hydrophobic chromatography.

6. The method for preparing a stable V-type 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 a stable V-type recombinant collagen according to claim 5, characterized in that, In step three, the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell.

8. The use of a stable V-type recombinant collagen prepared by the method according to any one of claims 5-7 in the preparation of collagen-containing products, said products being cosmetics or pharmaceuticals.

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