A method and application for transient expression of collagen in plants

Abstract

This invention provides a method and application for transient expression of collagen in plants. The collagen fragment C3S1, C3S2, C3S3, C3S4, and C3S5 are linked together with the amino acid sequences shown in SEQ1, SEQ2, SEQ3, SEQ4, and SEQ5, respectively. This collagen fragment can be expressed using tobacco as a bioreactor. By designing expression vectors, its expression effect in tobacco can be compared, and a transient expression system in tobacco can be established to achieve efficient expression of the collagen fragment in tobacco. This preparation method can express recombinant collagen fragments in tobacco with high expression levels. Compared to full-length intron-modified collagen or expression using type I collagen, this method achieves efficient expression of type III collagen fragments in tobacco bioreactors. It has a short cycle time, requires no maintenance of cell lines and bioreactors, significantly reduces production costs, and has a high safety index.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically involving an innovative technology for expressing recombinant collagen through plant bioreactors, and particularly involving a method for efficiently preparing bioactive type III collagen fragments C3S1-C3S5 using a tobacco transient expression system and its application in the fields of anti-oxidation and cell repair. Background Technology

[0002] Collagen, a major component of the extracellular matrix of mammalian cells, possesses a unique triple-helix structure and is rich in Gly-XY repeat sequences, demonstrating irreplaceable functional value in tissue repair, biomaterial construction, and regenerative medicine. However, traditional collagen extraction processes rely on animal tissues (such as bovine Achilles tendon and porcine skin), facing bottlenecks such as pathogen residue risks, poor batch-to-batch stability, and ethical controversies. Furthermore, the high molecular weight (approximately 300 kDa) and low solubility of natural collagen limit its application in targeted delivery and transdermal absorption. To address these challenges, recombinant collagen technology has emerged, using genetic engineering to express specific collagen fragments in microorganisms, mammalian cells, or plants, enabling efficient, safe, and customizable production.

[0003] In recent years, plant bioreactors have become a cutting-edge field in recombinant protein production due to their advantages of low cost, high scalability, and natural avoidance of animal-derived contamination risks. Among them, tobacco (Nicotiana abacum) is widely used in recombinant protein expression systems due to its short growth cycle (6-8 weeks), large biomass (over 100 tons of leaves per hectare), high genetic transformation efficiency, and non-competitive nature with the food chain. For example, studies have successfully used tobacco to express medical proteins such as antibodies, vaccine antigens, and growth factors. However, applying tobacco to the production of recombinant collagen fragments still faces multiple technical challenges.

[0004] Currently, optimization strategies for plant-derived recombinant collagen focus on gene design optimization and host modification. For example, patent (CN116970070B) increases the expression level of recombinant collagen in tobacco by adding introns, but it uses plant genetic transformation technology to express full-length collagen, which is time-consuming and involves complex purification processes. Although patent (CN118109506A) successfully produced type I collagen using BY-2 cells, significantly shortening the production cycle, it requires maintaining the cell line and bioreactor, necessitating substantial time and cost for maintenance.

[0005] Transient plant expression technology is a biomanufacturing method for rapidly producing recombinant proteins from living or ex vivo plant tissues. Its core lies in the transient introduction of exogenous genes into plant cells via non-integrative gene delivery systems (such as Agrobacterium-mediated infiltration, viral vectors, or nanocarriers). The target protein is then efficiently expressed within days using the host cell's transcription and translation mechanisms, without the need for stable genetic transformation or the production of transgenic plants. Due to its rapid response capabilities (e.g., the rapid production of candidate vaccine antigens from Nicotiana benthamiana leaves during the COVID-19 pandemic), low production costs (utilizing plant biomass as a natural bioreactor), and safety advantages (no risk of animal pathogen contamination), transient plant expression is gradually becoming an alternative production platform.

[0006] The application of plant transient expression technology to collagen production still faces the following technical bottlenecks:

[0007] Full-length collagen has a large molecular weight, making it difficult to express efficiently in plant systems; collagen is highly hydrophilic and easily forms inclusion bodies; transient expression systems have a short protein accumulation window. Summary of the Invention

[0008] Based on this, the present invention has successfully achieved efficient transient expression of five collagen fragments in tobacco by innovatively designing type III collagen functional fragments and optimizing expression vectors. Among them, the expression levels of C3S2 and C3S5 are significantly better than those of existing technologies, and their biological activity has been verified.

[0009] A recombinant type III collagen fragment, selected from: a) C3S1 protein with the amino acid sequence shown in SEQ 1; b) C3S2 protein with the amino acid sequence shown in SEQ 2; c) C3S3 protein with the amino acid sequence shown in SEQ 3;

[0010] d) C3S4 protein with the amino acid sequence shown in SEQ 4; e) C3S5 protein with the amino acid sequence shown in SEQ 5.

[0011] Accordingly, the present invention provides a tobacco transient expression system for efficient expression of recombinant proteins, comprising the plant expression vector pFolia40108, which uses a strong 35S promoter and a NOS terminator.

[0012] This carrier was previously disclosed in patent application document (CN106566842B).

[0013] Accordingly, the present invention also provides a method for constructing a transient expression vector for collagen fragments in tobacco, comprising the following steps:

[0014] A1: Synthesize gene fragments C3S1, C3S2, C3S3, C3S4, and C3S5;

[0015] A2: PCR amplification was performed using primers F-C3S1 / R-C3S1, F-C3S2 / R-C3S2, F-C3S3 / R-C3S3, F-C3S4 / R-C3S4, and F-C3S5 / R-C3S5.

[0016] A3: Add the amplified C3S1-C3S5 genes to the enzyme digestion system and digest with XhoI and BamHI;

[0017] A4: Perform agarose gel electrophoresis on the enzyme digestion products and recover the fragments to obtain purified DNA fragments;

[0018] A5: The recovered enzyme digestion products were mixed with the vector pFolia40108 digested with the same enzyme, and ligated with T4 DNA ligase to obtain recombinant plasmids.

[0019] A6 transformed the recombinant plasmid into E. coli DH5α competent cells and screened them on LB plates containing kanamycin;

[0020] A7: Select a single colony for amplification, extract the recombinant plasmid, and verify its correctness through sequencing.

[0021] Accordingly, the present invention also provides a method for transient expression of recombinant proteins C3S1, C3S2, C3S3, C3S4, and C3S5, comprising the following steps:

[0022] B1: The prepared recombinant plasmid was transformed into Agrobacterium tumefaciens strain C58C1 by heat shock; this strain is a commonly used Agrobacterium.

[0023] B2: Transfect the bacterial solution into tobacco leaves by injecting a sterile syringe;

[0024] B3: Quantitative analysis by Western blot: Expression levels from highest to lowest are C3S5 > C3S2 > C3S1 > C3S3 > C3S4. Preferred methods for purifying C3S2 and C3S5 collagen fragments include:

[0025] C1: Cation exchange chromatography: Cytiva SP Sepharose Fast Flow packing material was used, with equilibration buffer of 20 mM sodium acetate (pH 4.5) and elution gradient of 0-500 mM NaCl; C2: Anion exchange chromatography: Cytiva Q Sepharose Fast Flow packing material was used, with equilibration buffer of 20 mM Tris-HCl (pH 8.0) and elution gradient of 0-300 mM NaCl; C3: Concentration: The eluent was placed in a 10 kDa (1±20%) Da ultrafiltration centrifuge tube and centrifuged at 4 (1±20%) °C at 5000 (1±20%) × g for 15 (1±20%) minutes. The final protein concentration was in the range of 5.3~7.4 mg / mL.

[0026] This invention also provides the use of recombinant C3S2 and C3S5 proteins in promoting cell adhesion.

[0027] This invention also provides the application of C3S2 and C3S5 recombinant proteins in antioxidant preparations.

[0028] This invention also provides the application of recombinant C3S2 and C3S5 proteins in cell repair.

[0029] The beneficial effects of this invention are as follows:

[0030] This invention focuses on using tobacco plants as bioreactors and designs two types of expression vectors to compare their expression effects in tobacco plants. At the same time, a transient expression system for tobacco plants is established to achieve efficient expression of fragment collagen in tobacco.

[0031] This invention improves the mRNA stability of the type III collagen gene by adding multiple introns and signal peptides to express an apoplast, thereby increasing the expression level of plant-derived type III collagen and reducing the risk of collagen purification.

[0032] This invention expresses type III collagen in an apoplast, simplifying the purification process, reducing costs, and the addition of introns significantly enhances the stability of collagen expression. Attached Figure Description

[0033] Figure 1 This is a hydrophobicity analysis diagram of the sequence;

[0034] Figure 2 This is a protein selection fragment diagram;

[0035] Figure 3 This is an agarose gel electrophoresis image; M represents the D2000 Marker image.

[0036] Figure 4This is a diagram of a recombinant plasmid design pattern;

[0037] Figure 5 This is a PCR gel electrophoresis image; M represents the D2000 Marker image.

[0038] Figure 6 This is a Western blot analysis chromatogram of the recombinant protein extract;

[0039] Figure 7 This is an antioxidant analysis diagram. A represents DPPH free radicals, and B represents hydroxyl free radicals.

[0040] Figure 8 This is a diagram of cell adhesion analysis;

[0041] Figure 9 This is a cytotoxicity analysis diagram. a represents the cytotoxicity analysis of C2S2, and b represents the cytotoxicity analysis of C2S5.

[0042] Figure 10 These are images of bioelectron microscopy results. Image a represents the bioelectron microscopy result of C3S2, and image b represents the bioelectron microscopy result of C3S5. Detailed Implementation

[0043] Example 1: Design and Construction Strategy of Type III Collagen Fragments

[0044] (1) Hydrophilic sites

[0045] Literature review and analysis by the technology company indicate that fragments with higher hydrophilicity exhibit higher expression efficiency. Furthermore, to avoid potential restriction enzyme sites, isomerization sites (deamidation), degradation, and instability, this experiment selected hydrophilic and hydrophobic sites from the type III collagen fragment sequence for design. The hydrophilicity analysis process is as follows: The natural type III sequence (GenBank: AGL34959.1) was submitted to ProtScale (https: / / web.expasy.org / protscale / ) for hydrophobicity analysis. The sequence hydrophobicity analysis results are as follows. Figure 1 As shown in the figure. The results indicate that type III collagen has strong hydrophilicity. To further reduce selectivity and avoid duplication with other experiments, fragments with selection scores less than -0.75 were selected for subsequent analysis.

[0046] Table 1 Sequence Analysis

[0047]

[0048] Considering the final target fragment length is 30-60 kDa, or approximately 300-600 amino acids, the above sequences were combined. Using -1 as the mean threshold, the sequences can be divided into two design combinations. The aim was to achieve a uniform distribution, covering as much collagen region as possible. Except for the signal peptide tail, KEX2 (KR / RR / PR), YPS1 (RK / RR), and PEP4 (Leu-Leu- / -Val-Tyr) restriction sites were avoided, and the ASN and SER residues were minimized. The selected fragments are as follows... Figure 2 As shown, the final obtained sequence fragment is as follows:

[0049] Greater than -1: B,C1,C2,D1,E1,E2,F1,F2,G1,I1,K,L1,N

[0050] Less than -1: H1, H2, M1, O1, P1, P2

[0051] By arranging the above fragments in sequence, we can obtain the final sequence to be expressed. The first round can be defined as follows:

[0052] SEQ1: B+C1+C2+D1+E1+E2+F1+F2+G1+I1+K+L1+N (C3S1)

[0053] SEQ2: H1+H2+M1+O1+P1+P2 (C3S2)

[0054] SEQ3: B+C2+E1+ F1+G1+K+N (C3S3)

[0055] SEQ4: C1+D1+E2+F2+I1+L1 (C3S4)

[0056] SEQ5: B~P2 (C3S5)

[0057] Example 2: Construction of a transient expression vector for collagen fragments in tobacco

[0058] (1) Cloning of the target gene

[0059] The gene fragments C3S1, C3S2, C3S3, C3S4, and C3S5 synthesized in Example 1 were amplified by PCR using primers F-C3S1, R-C3S1, F-C3S2, R-C3S2, F-C3S3, R-C3S3, F-C3S4, and R-C3S4. The sequences of the upstream primer F and the downstream primer R are shown in Table 1.

[0060] Table 1 Primer Sequences

[0061]

[0062] The amplification products were identified by agarose gel electrophoresis, and the results are as follows: Figure 3 As shown in the figure. The results indicate that the size of the C3S1 amplification product is 1262 bp, the size of the C3S2 amplification product is 417 bp, the size of the C3S3 amplification product is 975 bp, the size of the C3S4 amplification product is 705 bp, and the size of the C3S5 amplification product is 318 bp, which are consistent with the target bands of the target genes C3S1, C3S2, C3S3, C3S4, and C3S5.

[0063] The amplified C3S1, C3S2, C3S3, C3S4, and C3S5 genes were added to the enzyme digestion system and digested. The reaction was carried out at 37°C for 2 h. The enzyme digestion system is shown in Table 2. The enzyme digestion products were recovered by agarose gel electrophoresis.

[0064] Table 2 Enzyme digestion reaction system

[0065]

[0066] Note: Reaction procedure: 37℃ for 2 hours. Enzyme digestion products were detected by agarose gel electrophoresis. BsmBI is a Type II restriction endonuclease that recognizes non-palindromic sequences and performs cleavage outside of the recognition sequence. The recognition sequences of BsmBI are 5' GGTCTC 3' and 3' CCAGAG 5'.

[0067] (3) Target fragment retrieval

[0068] The enzyme digestion products were subjected to agarose gel electrophoresis. The electrophoretic products were placed under UV light, and the target size bright band was excised along the edge as much as possible and placed in a 1.5 mL centrifuge tube. The fragments were recovered according to the steps and instructions of the Tiangen Agarose Gel DNA Recovery Kit to obtain purified DNA fragments.

[0069] (4) Ligation and transformation of enzyme digestion products to prepare recombinant plasmids

[0070] While the term "plasmid" in biology typically refers to a circular DNA molecule that replicates independently of chromosomal DNA in bacterial cells, the broader field of genetic engineering and molecular biology uses the term "vector" to describe any DNA molecule used for cloning and expressing genes, including vectors already used in plant and animal cells. This embodiment uses a plant expression vector, pFolia40108 (considered a special type of plasmid), as a vector to prepare a recombinant plasmid to improve the expression efficiency of exogenous genes in plants. The design pattern of the recombinant plasmid is shown in the diagram below. Figure 4 As shown.

[0071] The recovered enzyme digestion products were mixed with the vector pFolia40108, and T4 DNA ligase and 10× Buffer were added. The reaction system is shown in Table 3.

[0072] Table 3 Carrier Connection System

[0073]

[0074] Note: The GB system was used, and the reaction procedure was: ligation at 16℃ to obtain the recombinant plasmid.

[0075] The obtained recombinant plasmid was transferred into E. coli. The competent cells DH5α were taken out of the -80℃ freezer and thawed on ice. 5 μL of recombinant plasmid was added to 70 μL of competent cells DH5α, and the cells were repeatedly pipetted 5-8 times. The cells were then incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 90 s and immediately placed on ice for 3 min. 800 μL of LB medium equilibrated to room temperature was added, and the cells were cultured at 200 r / min and 37℃ with shaking for 1 h. The culture of the competent cells was centrifuged at 6000 r / min for 3 min, the supernatant was discarded, and the cells were resuspended in 100-120 μL of the remaining medium. 50 μL of the bacterial culture was evenly spread on a kanamycin LB agar plate and incubated upside down in a 37℃ incubator overnight.

[0076] (5) Extraction of recombinant plasmids

[0077] Single colonies were picked from LB agar plates and transferred to 5 mL LB liquid medium (containing 50 mg / mL Amp). The culture was incubated overnight at 37°C with shaking. In a sterile environment under laminar flow hood, five single colonies were picked using a sterile toothpick. The end of the toothpick containing the colony was then touched to the bottom of a PCR tube to leave a small amount of colony. This was used for colony PCR detection and subsequent culture expansion. The PCR reaction system is shown in Table 5.

[0078] Table 5 PCR detection reaction system

[0079]

[0080] Note: A 10 μL system was used, and the reaction program was 2×Taq: 94℃ deformation, 30 cycles, and 72℃ extension.

[0081] PCR products were detected by agarose gel electrophoresis, and the results are as follows: Figure 5As shown. E. coli transformed with pFolia40108-C3S1 showed a clear band at 1262 bp when identified with C3S1 primers; E. coli transformed with pFolia40108-C3S2 showed a clear band at 417 bp when identified with C3S2 primers; E. coli transformed with pFolia40108-C3S3 showed a clear band at 975 bp when identified with C3S3 primers; E. coli transformed with pFolia40108-C3S4 showed a clear band at 705 bp when identified with C3S4 primers; and E. coli transformed with pFolia40108-C3S5 showed a clear band at 318 bp when identified with C3S4 primers. A clear band was visible at the bp position; the gene fragments of the five recombinant proteins were consistent with the expected gene fragments. The recombinant plasmids were extracted from the bacterial culture of colonies with bands in the correct position by shaking, following the instructions provided by the plasmid mini-prep kit from Tiangen Pharmaceuticals.

[0082] (6) Sequencing

[0083] The recombinant plasmid was identified by PCR and sequencing. Sequencing was performed by Changsha Qingke Biotechnology Co., Ltd., and the obtained sequences were compared and analyzed with the target fragment using Snapgene software.

[0084] Example 2: Transient expression experiment of recombinant proteins C3S1, C3S2, C3S3, C3S4, and C3S5

[0085] (1) Transformation of Agrobacterium with recombinant plasmids

[0086] Recombinant plasmids C3S1, C3S2, C3S3, C3S4, and C3S5 were transformed into Agrobacterium tumefaciens strain C58C1 via heat shock transformation. Competent Agrobacterium cells were harvested and thawed on ice for 30 min. Approximately 1-2 μg of each of the C3S1, C3S2, C3S3, C3S4, and C3S5 recombinant plasmids were added to 100 μl of competent Agrobacterium cells and mixed thoroughly. The mixture was incubated on ice for 30 min, flash-frozen in liquid nitrogen for 1 min, then incubated in a 37°C water bath for 5 min, followed by an ice bath for 2 min. 800 μl of liquid LB medium was added, and the mixture was incubated at 28°C and 200 rpm for 2 h. The bacterial culture was centrifuged at 6000 rpm for 3 min, the medium was removed to a final volume of 100 μL, and the culture was gently mixed by pipetting. The culture was then spread onto solid LB culture dishes containing Rif and Carb, and incubated at 28°C for 48 h. After the colonies have grown plaques, select suitable single colonies in a sterile environment under a laminar flow hood. Confirm successful transformation with positive PCR testing of the colonies, then propagate the bacterial culture. Aliquot the culture into 1.5 mL centrifuge tubes, add 60% glycerol, and store at -80°C for subsequent tobacco transfection.

[0087] (2) Injection transfection of tobacco and sampling

[0088] ① Solution preparation:

[0089] LB medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, and ddH2O to make up the volume.

[0090] 0.1M MES (pH 5.6) (1 L): 19.524 g MES, pH adjusted to 5.6 with NaOH, volume made up with ddH2O, filtered through a 0.22 μm filter for sterilization, and stored at room temperature.

[0091] 100 mM acetylsyl syringone (AS): 0.036 g acetylsyl syringone dissolved in 1 mL dimethyl sulfoxide (DMSO).

[0092] 1M MgCl2 (400 mL): 95.2 g MgCl2·H2O, add ddH2O to make up the volume, and store at room temperature.

[0093] Transfection solution: 10 mM MES, 100 μM AS, 10 mM MgCl2, ddH2O to make up the volume.

[0094] ② Injecting tobacco

[0095] Agrobacterium containing the target plasmid was cultured by shaking, and approximately 5 mL of bacterial suspension was collected. The bacterial pellet was collected by gentle centrifugation (room temperature, 6000 rpm, 5 min). The colonies were resuspended in transfection buffer and activated by shaking at 28°C and 100 rpm for 1 h. The OD values ​​of the three activated bacterial suspensions were adjusted to 0.1. The bacterial suspension was then injected into the leaves using a sterile syringe for transfection. The transfected tobacco leaves were photographed and sampled over the next two weeks.

[0096] (3) Western blot analysis

[0097] On day 7 post-transfection, 0.2 g of tobacco leaves containing the expressed fraction were placed in a 1.5 mL centrifuge tube, flash-frozen in liquid nitrogen, and ground into powder using a grinder. 500 μL of NP-40 lysis buffer was added to the centrifuge tube to resuspend the leaf powder. The tube was incubated on ice for 10 min, then centrifuged at 14000 rpm for 15 min at 4°C. The supernatant was collected for Western blot analysis. Figure 6 As shown in the figure. The results showed that the expression levels from largest to smallest were C3S5>C3S2>C3S1>C3S3>C3S4, and the band positions of C3S1 to C3S5 were 46.3 kDa, 15.2 kDa, 35.8 kDa, 25.8 kDa, and 68.1 kDa, respectively.

[0098] (4) Real-time quantitative PCR (qRT-PCR) analysis

[0099] Primers were designed using Primer 5 software (Table 6) and synthesized by Qingke Biotechnology Co., Ltd. RNA from five genes was extracted using the Omega R6827 kit, and cDNA obtained by reverse transcription (Vazyme kit) was used as a template. The samples were added according to the real-time quantitative PCR reaction system (Table 7), and after membrane bonding and centrifugation, qRT-PCR amplification was performed.

[0100] like Figure 7 As shown, compared with the unexpressed recombinant collagen, all five recombinant collagen proteins were stably expressed in tobacco, and the expression levels, from highest to lowest, were C3S5>C3S2>C3S1>C3S3>C3S4, consistent with the results of Western blot analysis. Since C2S5 and C2S2 had high and similar expression levels, C3S5 and C3S2 were selected for subsequent experiments.

[0101] Table 6 qRT primer sequences

[0102]

[0103] Table 7 qPCR detection reaction system

[0104]

[0105] Example 3: Purification experiment of recombinant proteins C3S5 and C3S2

[0106] (1) Solution preparation

[0107] Plant cell lysis buffer: 20 mM Tris-HCl, 4.8 mM mercaptoethanol (β-ME), 1 mM benzosulfonyl fluoride (PMSF).

[0108] Equilibrium solution: 20 mM Tris-HCl (pH 8.0);

[0109] Nonspecific protein elution buffer (WB): 50 mM NaCl, 20 mM Tris-HCl (pH 8.0).

[0110] Protein elution buffer EB: 300 mM NaCl, 20 mM Tris-HCl (pH 8.0);

[0111] Dialysis fluid: 20 mM Tris-HCl (pH 8.0);

[0112] Anion exchange chromatography packing material: Cytiva Q Sepharose Fast Flow packing material was used, with equilibration buffer of 20 mM Tris-HCl (pH 8.0) and elution gradient of 0-300 mM NaCl;

[0113] Cation exchange chromatography packing material: Cytiva SP Sepharose Fast Flow packing material was used, with equilibration buffer of 20 mM sodium acetate (pH 4.5) and elution gradient of 0-500 mM NaCl.

[0114] (2) Extraction of crude extract

[0115] Tobacco leaves transfected with Agrobacterium and exhibiting a distinct phenotype were harvested, and the main veins of the leaves were removed. One kg of tobacco leaf containing C3S5 collagen fragments was weighed and mixed in a cryoextraction buffer of 20 mM Tris-HCl, 4.8 mM β-ME, and 1 mM benzosulfonyl fluoride (PMSF). 15 g of polyvinylpyridinium (PVPP) and 6 g of activated charcoal were then added. The mixing was performed five times, one minute each, while maintaining the temperature below 15°C. The crude extract was filtered through a gauze pad, centrifuged for 30 min at 26000 g and 5 °C; the supernatant was collected, and CaCl2 was added to a final concentration of 10 mM, and activated carbon concentration was 1 g / L; 5 mg / L ficin (Sigma # F4125; 15 °C, stirred for 3 h) was added; insoluble contaminants were removed by centrifugation (30 min, 22000 g, 15 °C); the C3S5 fragment collagen in the recovered supernatant was gradually added to crystalline NaCl, precipitating to a final concentration of 3.13 M (25 min, rt, continuously stirred); the solution was incubated in a refrigerator for 8 hours, and then centrifuged (26000 g, 2 h, 5 °C) to collect particles containing C3S5 fragment collagen. The extraction method for C3S2 fragment collagen particles was the same as above.

[0116] (3) Purification of C3S5 and C3S5 fragment collagen

[0117] ② Cation exchange chromatography:

[0118] Separation and purification of C3S5 fragment collagen was achieved under low-temperature conditions using the charge interaction between the collagen and the cation exchange medium. 15 mL of SPFF packing material was pipetted into a 50 mL gravity column, allowing the medium to settle freely and the stock solution to be drained. The packing material was then compacted using a 20-micron circular sieve plate to stabilize it. 100 mL of equilibration buffer was added to the column, allowing it to flow through. Note that the packing material should not be dried out; a small amount of liquid should remain on the upper sieve plate to complete equilibration. The supernatant from approximately 50 mL of the crude C3S5 fragment collagen extract was centrifuged and filtered through a 0.22-micron filter membrane to obtain the sample. This sample was then slowly added to the column, allowing gravity to allow the liquid to flow out, thus completing the sample loading process.

[0119] ③ Rinsing:

[0120] The column was rinsed with 100 mL of buffer solution (dialysis fluid) and then the packing material was rinsed with 100 mL of nonspecific protein elution buffer (WB).

[0121] ⑤ Washing off:

[0122] C3S5 collagen fragments were eluted using 50 mL of specific protein elution buffer EB added to a gravity column, and the eluent was collected in a 50 mL centrifuge tube.

[0123] Anion exchange chromatography:

[0124] Separation and purification of C3S5 fragment collagen and anion exchange medium were achieved under low-temperature conditions. An appropriate amount of QSFF packing material was placed in a gravity column, allowing the medium to settle freely and the stock solution to be drained. The packing material was then compacted with a 20-micron circular sieve plate to stabilize it. 100 mL of equilibration buffer was added to the column, and the material was allowed to flow through. Note that the packing material should not be dried out; therefore, a small amount of liquid should remain on the upper sieve plate to complete equilibration. Approximately 50 mL of the eluent obtained from cation chromatography was filtered through a 0.22-micron filter membrane and then directly added to the anion exchange chromatography column. The flow-through was collected using a centrifuge tube.

[0125] ⑥ Dialysis:

[0126] Cut the dialysis bag to an appropriate length, with a molecular weight cutoff of 10 kDa. After cleaning and leak testing the dialysis bag, place the anion-collected flow-through solution into 500 mL of pre-cooled dialysis solution and perform dialysis overnight at 4°C. Change the dialysis solution every 12 hours, for a total of two changes.

[0127] ⑦ Concentrate:

[0128] Using a 50 mL ultrafiltration tube with a 5 kDa capacity, pre-cool the tube on ice for a few minutes, and add 10-12 mL of the dialyzed sample to the upper part of the tube. Balance the ultrafiltration tube to ensure that both the mass and center of gravity are in equilibrium, and centrifuge at 5000×g for 15 min at 4°C using a high-speed refrigerated centrifuge. Repeat the centrifugation 2-4 times until the purified C3S5 fragment collagen sample is concentrated to approximately 1 mL.

[0129] The purification method for C3S2 fragment collagen flake particles is the same as above.

[0130] Example 4: Functional detection of recombinant proteins C2S2 and C2S5

[0131] (1) Antioxidant activity test

[0132] According to Xin Xuanying's method (Xin Xuanying, Li Meiyao et al. 2023), the antioxidant activity of collagen was determined with vitamin C and butylated hydroxyanisole as control groups. The in vitro antioxidant effect was explored by measuring the scavenging capacity of DPPH free radicals and hydroxyl free radicals.

[0133] DPPH free radicals: Take 5 colorimetric tubes and add 3 mL of 0.05 mmol / L DPPH solution, then add 3 mL of collagen solution at concentrations of 0.5, 1.0, 1.5, 2.0, and 3.0 mg / mL, respectively. Shake thoroughly, let stand for 30 min, and measure the absorbance (A1) at a wavelength of 517 nm. A control group (A2) is prepared by mixing 3 mL of anhydrous ethanol with the gradient solution, and a blank group (A0) is prepared by mixing 3 mL of anhydrous ethanol with 3 mL of DPPH solution. The DPPH free radical scavenging rate is calculated as: Scavenging rate X1 = (1 - (A1 - A2) / A0) × 100%.

[0134] Hydroxyl radicals: 1 mL of 0.025 mol / L phosphate buffer (pH=7.4), 1 mL of 40 μg / mL safranin solution, 0.5 mL of collagen solutions at concentrations of 0.5, 1.0, 1.5, 2.0, and 3.0 mg / mL, 1 mL of 0.945 mmol / L ferric sodium EDTA, and 1 mL of 3% hydrogen peroxide solution were mixed and heated in a water bath at 37℃ for 30 min. The absorbance A1 was measured at a wavelength of 520 nm. The blank group A0 consisted of 0.5 mL of distilled water, and the control group consisted of 1.5 mL of distilled water. The scavenging rate of hydroxyl radicals was calculated as: Scavenging rate X2 = (A1-A0) / (A2-A0) × 100%.

[0135] The results showed that within the range of 0.5-3.0 mg / mL, the higher the concentration of collagen solution, the stronger the antioxidant effect. Specifically, C3S5 exhibited scavenging rates of 82.5% and 42.3% for DPPH and hydroxyl radicals, respectively; while C3S2 showed scavenging rates of 80.4% and 41.2% for DPPH and hydroxyl radicals, respectively.

[0136] (2) Cell adhesion detection

[0137] Following and modifying the method of Kang (Kang, Wang et al. 2018), 0.5 mg / mL recombinant collagen solution was added to 96-well cell culture plates. The control group consisted of heat-denatured 10% bovine serum albumin (BSA) solution and collagen standard. After incubation at 4°C for 24 h, the supernatant was discarded. 100 μL of L929 suspension was added, and the cells were cultured at 37°C for 6–9 h. Adhesive cells were lysed by total DNA quantification using ultrapure water through three freeze-thaw cycles. 5 µg / mL Hochest 33258 was added to the resulting cell lysate, and the cells were incubated in the dark for 1 h. Absorbance values ​​were read using a microplate reader at 360 nm excitation and 465 nm emission wavelengths. Each sample was measured in triplicate, and the data are expressed as mean ± standard deviation (SD).

[0138] like Figure 8 As shown, the adhesion percentages of C3S2 and C3S5 were 120.42% and 130.43%, respectively, both higher than those of the collagen standard and BSA protein. This indicates that C3S2 and C3S5 are more readily absorbed and utilized by L929 cells than the collagen standard. Adding C3S2 and C3S5 can significantly promote L929 cell adhesion, enhance the specific binding of cells to adjacent cells or the extracellular matrix, and facilitate cell regulation, demonstrating potential for application in skin fibroblast repair.

[0139] (3) Cytotoxicity detection

[0140] Following the method of He Huixia (He Huixia, 2023), the toxicological effects of different concentrations of collagen solutions on L929 cells were systematically evaluated using the Cell Counting Kit-8 (CKK-8) method to clarify its biocompatibility. Solutions with recombinant collagen concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.3 mg / mL were prepared, filtered through a 0.22 μm filter membrane, and stored at 4 ℃. 100 μL of L929 suspension (5 × 10⁻⁶ cells / well) was added to each 96-well cell culture plate. 6Cells (cells / mL) were cultured in a CO2 incubator at 37 ℃ and 5% CO2 for 24 h, then the culture medium was discarded. 100 μL of recombinant collagen solutions of different concentrations were added. A control group was prepared with 100 μL of DMEM medium. After 24 h of culture, the supernatant was discarded. 100 μL of CKK-8 cell culture medium containing 10% CO2 was added to each sample well and control well. After culturing in the cell incubator for 2 h, the absorbance at 450 nm was measured using a microplate reader. Each sample was measured in triplicate. The absorbance value of the sample group is A. t The absorbance value of the control group is A. c The absorbance value of the blank group is A0. The formula for calculating cell viability (A...) t -A0) / (A c -A0)×100%.

[0141] The results showed that within the concentration range (0.01-0.3 mg / L), the cell viability of C3S2 and C3S5 gradually increased with increasing concentration (e.g., ...). Figure 9 Among them, C3S2 activity reached a maximum of 120.3%, and C3S5 activity reached a maximum of 135.5%. This indicates that these two proteins can effectively enhance the activity of organisms and do not produce toxicity to L929 cells.

[0142] (4) Biological scanning electron microscopy (SEM) detection

[0143] Following the method of Marin (Marin, Albu Kaya et al. 2018), recombinant collagen (50 µL, concentration 1 mg / mL) was mixed with 5 µL of fibroblast buffer (200 mM Na2HPO4, pH 11.2) and incubated at 37°C for 1 h to induce fibroblast formation. The fibrils were collected by centrifugation (5 min, 13000 rpm). The collagen fibrils samples were then immersed in 0.1 M phosphate buffer (pH 7.2) and 2.5% glutaraldehyde, and rinsed 5 times in phosphate buffer before being sent to the Scientific Compass Testing Platform (Changsha) for analysis.

[0144] The results showed that at 1 μm, C3S2 and C3S5 exhibited a fibrous structure (e.g., Figure 10 This demonstrates its important role in biomechanics, cell interaction, and tissue repair.

[0145] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A recombinant type III collagen fragment, characterized in that, Selected from: a) C3S1 protein with the amino acid sequence shown in SEQ 1; b) The C3S2 protein with the amino acid sequence shown in SEQ 2; c) The C3S3 protein with the amino acid sequence shown in SEQ 3; d) The C3S4 protein with the amino acid sequence shown in SEQ 4; e) C3S5 protein with the amino acid sequence shown in SEQ 5.

2. A method for constructing a tobacco transient expression vector for recombinant type III collagen fragments as described in claim 1, characterized in that, It includes the following steps: A1: Synthesize the nucleic acid gene fragments encoding the C3S1, C3S2, C3S3, C3S4, and C3S5 proteins; A2: Use primers F-C3S1 / R-C3S1, F-C3S2 / R-C3S2, F-C3S3 / R-C3S3, F-C3S4 / R-C3S4, and F-C3S5 / R-C3S5 to perform PCR amplification on each nucleic acid gene fragment obtained in step A1. A3: Add the amplified C3S1-C3S5 genes to the enzyme digestion system and digest with XhoI and BamHI; A4: Perform agarose gel electrophoresis on the enzyme digestion products and recover the fragments to obtain purified DNA fragments; A5: The recovered enzyme digestion products were mixed with the vector pFolia40108 digested with the same enzyme, and ligated with T4 DNA ligase to obtain recombinant plasmids. A6 transformed the recombinant plasmid into E. coli DH5α competent cells and screened them on LB plates containing kanamycin; A7: Select a single colony for amplification, extract the recombinant plasmid, and verify its correctness through sequencing.

3. A method for transient expression of recombinant proteins C3S1, C3S2, C3S3, C3S4, and C3S5 as described in claim 1, characterized in that, It includes the following steps: B1: The recombinant plasmid prepared according to claim 2 is transformed into Agrobacterium tumefaciens strain C58C1 by heat shock; B2: Transfect the bacterial solution into tobacco leaves by injecting a sterile syringe; B3: Quantitative analysis by Western blot: The expression levels from highest to lowest are C3S5>C3S2>C3S1>C3S3>C3S4.

4. The method as described in claim 3, characterized in that, C3S2 and C3S5 fragment collagen purification methods include: C1: Cation exchange chromatography: Cytiva SP Sepharose Fast Flow packing material was used, with equilibration buffer of pH 4.5 and 20 mM sodium acetate, and elution gradient of 0-500 mM NaCl; C2: Anion exchange chromatography: Cytiva Q Sepharose Fast Flow packing material was used, with equilibration buffer of pH 8.0 and 20 mM Tris-HCl, and elution gradient of 0-300 mM NaCl; C3: Concentration. Place the eluent in an 8–12 kDa ultrafiltration centrifuge tube and centrifuge at 4000–6000 × g for 12–18 minutes at 3.2–4.8 °C. The final protein concentration is in the range of 5.3–7.4 mg / mL.

5. Use of the C3S2 and C3S5 recombinant proteins as described in claim 1 in the preparation of articles that promote cell adhesion.

6. The application of the C3S2 and C3S5 recombinant proteins as described in claim 1 in the preparation of antioxidant agents.

7. The application of the C3S2 and C3S5 recombinant proteins as described in claim 1 in the preparation of cell repair agents.

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