Method for plant transient expression of collagen and application

Abstract

The invention provides a method for plant transient expression of collagen and application of the plant transient expression of the collagen, and fragment collagen C3S1, C3S2, C3S3, C3S4 and C3S5 is formed by combining and connecting amino acid sequences shown as SEQ1, SEQ2, SEQ3, SEQ4 and SEQ5 respectively. According to the fragment collagen, plant tobacco can be used as a bioreactor, an expression vector is designed, the expression effect of the fragment collagen in the plant tobacco is compared, and meanwhile, a plant tobacco transient expression system is established, so that efficient expression of the fragment collagen in the tobacco is realized. By adopting the preparation method, tobacco can be utilized to express fragment collagen recombinant protein, a relatively high expression quantity is obtained, and compared with transformation by using full-length added intron or expression by using I-type collagen, the method realizes efficient expression of fragment collagen in III-type collagen in plant bioreactor tobacco, the period is short, and the yield is high. Maintenance of a cell line and a bioreactor is not needed, the production cost is remarkably reduced, and the safety index is high.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to an innovative technology for expressing recombinant collagen through a plant bioreactor, and more particularly to 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 Art

[0002] As the main component of the extracellular matrix of mammals, collagen has a unique triple helix structure and is rich in Gly-XY repeat sequences. It shows irreplaceable functional value in the fields of tissue repair, biomaterial construction and regenerative medicine. However, the traditional collagen extraction process relies on animal tissues (such as bovine Achilles tendon, pig skin, etc.), and faces bottlenecks such as the risk of pathogen residues, poor stability between batches and ethical disputes. In addition, the high molecular weight (about 300kDa) and low solubility of natural collagen limit its application in scenarios such as targeted delivery and transdermal absorption. For this reason, recombinant collagen technology came into being, using genetic engineering to express specific collagen fragments in microorganisms, mammalian cells or plants to achieve efficient, safe and customizable production.

[0003] In recent years, plant bioreactors have become a cutting-edge direction in recombinant protein production due to their low cost, high scalability, and natural avoidance of animal-source contamination risks. Among them, tobacco (Nicotiana tabacum) is widely used in recombinant protein expression systems due to its short growth cycle (6-8 weeks), large biomass (leaf yield of more than 100 tons per hectare), high genetic transformation efficiency, and non-competition with the food chain. For example, studies have successfully used tobacco to express medical proteins such as antibodies, vaccine antigens, and growth factors. However, the application of tobacco in 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 of recombinant collagen in tobacco by adding introns. However, it uses plant genetic transformation technology to express full-length collagen, which requires a long production cycle and a complex purification process. Patent (CN118109506A) successfully produces type I collagen using BY-2 cells, significantly shortening the production cycle. However, it requires maintaining the cell line and bioreactor, which requires a lot of time and cost.

[0005] Plant transient expression technology is a biomanufacturing method for rapidly producing recombinant proteins in living plants or excised plant tissues. Its core approach involves transiently introducing exogenous genes into plant cells via non-integrating gene delivery systems (such as Agrobacterium infiltration, viral vectors, or nanocarriers). The target protein is then efficiently expressed within days using the host cell's transcriptional and translational machinery, eliminating the need for stable genetic transformation or the generation of transgenic plants. Plant transient expression, due to its rapid response, low production costs (utilizing plant biomass as a natural bioreactor), and safety advantages (no risk of contamination by animal pathogens), is becoming an alternative production platform.

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

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

[0008] Based on this, the present invention innovatively designed functional fragments of type III collagen and optimized the expression vector to successfully achieve efficient transient expression of five collagen fragments in tobacco. Among them, the expression levels of C3S2 and C3S5 were significantly better than those of the existing technology, and their biological activity was verified.

[0009] A recombinant type III collagen fragment selected from:

[0010] a) C3S1 protein having the amino acid sequence shown in SEQ 1;

[0011] b) C3S2 protein having the amino acid sequence shown in SEQ2;

[0012] c) C3S3 protein having the amino acid sequence shown in SEQ 3;

[0013] d) C3S4 protein having the amino acid sequence shown in SEQ 4;

[0014] e) C3S5 protein with the amino acid sequence shown in SEQ 5.

[0015] Accordingly, the present invention provides a tobacco transient expression system for efficient expression of recombinant proteins, comprising a plant expression vector pFol ia40108, which uses a strong 35S promoter and a NOS terminator.

[0016] This carrier was previously disclosed in a patent application document (CN106566842B).

[0017] Accordingly, the present invention also provides a method for constructing a tobacco transient expression vector for collagen fragments, comprising the following steps:

[0018] A1: Synthetic gene fragments C3S1, C3S2, C3S3, C3S4, and C3S5;

[0019] A2: PCR amplification using primers F-C3S1 / R-C3S1, F-C3S2 / R-C3S2, F-C3S3 / R-C3S3, F-C3S4 / R-C3S4, and F-C3S5 / R-C3S5;

[0020] A3: Add the amplified C3S1-C3S5 genes to the enzyme digestion system and double-digest with XhoI and BamHI;

[0021] A4: Run the digestion product on agarose gel electrophoresis and recover the fragments to obtain purified DNA fragments;

[0022] A5: The recovered enzyme digestion products were mixed with the vector pFolia40108 digested with the same enzymes, and ligated with T4 DNA ligase to obtain recombinant plasmids.

[0023] A6 The recombinant plasmid was transformed into E. coli DH5α competent cells and screened on LB plates containing kanamycin;

[0024] A7: Pick a single colony for amplification, extract the recombinant plasmid and verify its correctness by sequencing.

[0025] Accordingly, the present invention also provides a method for transient expression of recombinant proteins C3S1, C3S2, C3S3, C3S4, and C3S5, comprising the following steps:

[0026] B1: The recombinant plasmid prepared in claim 3 is transformed into Agrobacterium tumefaciens C58C1 strain by heat shock; this strain is a commonly used Agrobacterium;

[0027] B2: Use a sterile syringe to inject the bacterial solution into tobacco leaves for transfection;

[0028] B3: Quantitative analysis by Western blot: the expression levels from high to low are C3S5>C3S2>C3S1>C3S3>C3S4.

[0029] Preferably, the method for purifying C3S2 and C3S5 fragment collagen comprises:

[0030] C1: Cation exchange chromatography: using Cytiva SP Sepharose Fast Flow filler, equilibration buffer: 20 mM sodium acetate (pH 4.5), elution gradient: 0-500 mM NaCl;

[0031] C2: Anion exchange chromatography: using Cytiva Q Sepharose Fast Flow filler, equilibration buffer: 20 mM Tris-HCl (pH 8.0), elution gradient: 0-300 mM NaCl;

[0032] C3: Concentration: Place the eluate in a 10k (1 ± 20%) Da ultrafiltration centrifuge tube and centrifuge at 5000 (1 ± 20%) × g for 15 (1 ± 20%) minutes at 4 (1 ± 20%) °C. The final protein concentration is in the range of 5.3 to 7.4 mg / mL.

[0033] The present invention also provides the use of C3S2 and C3S5 recombinant proteins in promoting cell adhesion.

[0034] The present invention also provides the use of C3S2 and C3S5 recombinant proteins in antioxidant preparations.

[0035] The present invention also provides the use of C3S2 and C3S5 recombinant proteins in cell repair.

[0036] The beneficial effects of the present invention are as follows:

[0037] The present invention focuses on using tobacco plants as bioreactors, designs two types of expression vectors, compares their expression effects in tobacco plants, and establishes a transient expression system in tobacco plants to achieve efficient expression of fragmented collagen in tobacco.

[0038] The present invention increases the expression level of plant-derived type III collagen by adding multiple introns and signal peptides to the apoplast to express the type III collagen gene, thereby improving the stability of mRNA and reducing the risk of purifying collagen.

[0039] The present invention expresses type III collagen in the apoplast, simplifies the purification steps, reduces costs, and significantly enhances the stability of collagen expression by adding intron optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the hydrophobicity analysis graph of the sequence;

[0041] Figure 2 It is a protein selection fragment map;

[0042] Figure 3 is an agarose gel electrophoresis image, M represents the D2000 Marker image;

[0043] Figure 4 This is a diagram of the recombinant plasmid design pattern;

[0044] Figure 5It is the PCR gel electrophoresis image, M represents the D2000 Marker image;

[0045] Figure 6 is a Western blot analysis of the recombinant protein extract;

[0046] Figure 7 This is an antioxidant analysis chart, A represents DPPH free radicals, B represents hydroxyl free radicals;

[0047] Figure 8 is a graph of cell adhesion analysis;

[0048] Figure 9 It is a cytotoxicity analysis diagram, a represents the cytotoxicity analysis of C2S2, and b represents the cytotoxicity analysis of C2S5;

[0049] Figure 10 These are the bio-electron microscopy results. a represents the bio-electron microscopy result of C3S2, and b represents the bio-electron microscopy result of C3S5. DETAILED DESCRIPTION

[0050] Example 1: Design and construction strategy of type III collagen fragments

[0051] (1) Hydrophilic sites

[0052] The analysis of literature references and technology companies shows that the more hydrophilic the fragment, the higher the expression efficiency. At the same time, in order to avoid potential enzyme cleavage sites, isomerization sites (deamidation), degradation and instability, this experiment selected the hydrophilic and hydrophobic sites of 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 is as follows Figure 1 The results showed that type III collagen was highly hydrophilic. To further narrow the selectivity and avoid duplication with other test contents, fragments with a score less than -0.75 were selected for subsequent analysis.

[0053] Table 1 Sequence analysis

[0054]

[0055] Considering that the length of the final target fragment is 30-60 kDa, that is, about 300-600 aa, the above sequences are combined. Taking the mean as -1 as the boundary, the above sequences can be divided into two types of design combinations. Considering the distribution as evenly as possible and covering more collagen regions for combination. In addition to the signal peptide tail, avoid KEX2 (KR / RR / PR), YPS1 (RK / RR), PEP4 (Leu-Leu- / -Val-Tyr) restriction sites, try to avoid ASN, SER two residues, and select fragments such as Figure 2 As shown, the final sequence fragments obtained are as follows:

[0056] Greater than -1: B,C1,C2,D1,E1,E2,F1,F2,G1,I1,K,L1,N,

[0057] Less than -1: H1, H2, M1, O1, P1, P2

[0058] By arranging the above fragments in sequence, the final sequence to be expressed can be obtained. The first round can be determined as follows:

[0059] SEQ1:B+C1+C2+D1+E1+E2+F1+F2+G1+I1+K+L1+N(C3S1)

[0060] SEQ2:H1+H2+M1+O1+P1+P2(C3S2)

[0061] SEQ3:B+C2+E1+F1+G1+K+N(C3S3)

[0062] SEQ4:C1+D1+E2+F2+I1+L1(C3S4)

[0063] SEQ5:B~P2(C3S5)

[0064] Example 2: Construction of tobacco transient expression vector for collagen fragment

[0065] (1) Target gene cloning

[0066] 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.

[0067] Table 1 Primer sequences

[0068]

[0069] The amplified products were identified by agarose gel electrophoresis. Figure 3 The results showed that the size of the amplified product of C3S1 was 1262 bp, the size of the amplified product of C3S2 was 417 bp, the size of the amplified product of C3S3 was 975 bp, the size of the amplified product of C3S4 was 705 bp, and the size of the amplified product of C3S5 was 318 bp, which were consistent with the target bands of the target genes C3S1, C3S2, C3S3, C3S4, and C3S5.

[0070] The amplified C3S1, C3S2, C3S3, C3S4 and C3S5 genes were added to the enzyme digestion system and digested at 37°C for 2 h. The enzyme digestion system is shown in Table 2, and the digestion products were recovered by agarose gel electrophoresis.

[0071] Table 2 Enzyme digestion reaction system

[0072]

[0073] Note: Reaction procedure: Incubate at 37°C for 2 hours. Digestion products were detected by agarose gel electrophoresis. BsmBI is a Type IIs restriction endonuclease that recognizes non-palindromic sequences and cleaves outside of the recognition sequence. The recognition sequences for BsmBI are 5'GGTCTC 3' and 3'CCAGAG 5'.

[0074] (3) Target fragment recovery

[0075] Run the digested product on agarose gel electrophoresis under UV light. Cut the desired bright band along the edge as closely as possible and place it in a 1.5 mL centrifuge tube. Recover the fragment according to the Tiangen Agarose Gel DNA Recovery Kit instructions to obtain purified DNA fragments.

[0076] (4) Ligation and transformation of enzyme-digested products to prepare recombinant plasmids

[0077] Although the term "plasmid" in biology usually refers to a circular DNA molecule that replicates independently of chromosomal DNA in bacterial cells, in the broader field of genetic engineering and molecular biology, the term "vector" can be used to describe any DNA molecule used to clone and express genes, including vectors used in plant and animal cells. This example uses a plant expression vector pFolia40108 (considered a special type of plasmid) as a vector to prepare a recombinant plasmid for improving the expression efficiency of foreign genes in plants. The recombinant plasmid design pattern is shown in the figure below. Figure 4 shown.

[0078] 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.

[0079] Table 3 Vector ligation system

[0080]

[0081] Note: GB system was used, reaction procedure: 16℃ ligation, recombinant plasmid was obtained

[0082] The obtained recombinant plasmid was transformed into Escherichia coli, and the competent cells DH5α were taken out from the -80℃ refrigerator and thawed on ice; 5 μL of the recombinant plasmid was added to 70 μL of competent cells DH5α, repeatedly pipetted 5 to 8 times, and incubated on ice for 30 minutes; heat shock was performed in a 42℃ water bath for 90 seconds, and immediately placed on ice for 3 minutes; 800 μL of LB medium equilibrated to room temperature was added, and shaking culture was carried out at 200 r / min and 37℃ for 1 hour; the above-cultured competent cell culture fluid was centrifuged at 6000 r / min for 3 minutes, the supernatant was discarded, and the bacteria were resuspended with the remaining 100 to 120 μL of culture medium, 50 μL of the bacterial liquid was evenly spread on the kanamycin LB agar plate, and inverted in a 37℃ incubator for overnight culture.

[0083] (5) Extraction of recombinant plasmid

[0084] Transfer a single colony from the LB agar plate to 5 mL of LB liquid medium (containing 50 mg / mL Amp) and culture with shaking at 37°C overnight. In the sterile environment of a clean bench, pick five single colonies with a sterile toothpick and touch the end of the toothpick with the colony to the bottom of a PCR tube to retain a small amount of colonies. Use this for colony PCR testing and expand the culture. The PCR reaction system is shown in Table 5.

[0085] Table 5 PCR detection reaction system

[0086]

[0087]

[0088] Note: A 10 μL system was used, and the reaction program was 2× Taq: 94°C denaturation, 30 cycles, and 72°C extension.

[0089] The PCR products were detected by agarose gel electrophoresis. Figure 5As shown. The E. coli transformed into pFolia40108-C3S1 showed a clear band at 1262 bp after identification with C3S1 primers; the E. coli transformed into pFolia40108-C3S2 showed a clear band at 417 bp after identification with C3S2 primers; the E. coli transformed into pFolia40108-C3S3 showed a clear band at 975 bp after identification with C3S3 primers; The E. coli transformed with pFolia40108-C3S5 showed obvious bands at 705bp when identified by C3S4 primers; the E. coli transformed with pFolia40108-C3S5 showed obvious bands at 318bp when identified by C3S4 primers; the gene fragments of the five recombinant proteins were consistent with the expected gene fragments, and the bacterial culture liquid of the colonies with bands and correct positions was selected for the extraction of recombinant plasmids, and the recombinant plasmids were extracted according to the instructions provided by Tiangen Company's plasmid mini-extraction kit.

[0090] (6) Sequencing

[0091] The recombinant plasmid was identified by PCR and sequencing. Sequencing was performed by Changsha Qingke Biotechnology Co., Ltd., and the obtained sequence was aligned with the target fragment using Snapgene software.

[0092] Example 2 Transient expression experiment of recombinant proteins C3S1, C3S2, C3S3, C3S4, and C3S5

[0093] (1) Transformation of Agrobacterium with recombinant plasmid

[0094] Transform the C3S1, C3S2, C3S3, C3S4, and C3S5 recombinant plasmids into Agrobacterium tumefaciens C58C1 strain via heat shock transformation. Remove the competent Agrobacterium and thaw on ice for 30 minutes. Add approximately 1-2 μg of the C3S1, C3S2, C3S3, C3S4, and C3S5 recombinant plasmids to 100 μl of competent Agrobacterium cells and mix thoroughly. Incubate on ice for 30 minutes, then quickly freeze in liquid nitrogen for 1 minute. Incubate in a 37°C water bath for 5 minutes, and then incubate on ice for 2 minutes. Add 800 μl of liquid LB medium and incubate at 28°C at 200 rpm for 2 hours. Centrifuge the culture at 6000 rpm for 3 minutes, remove the culture medium, and reduce the volume to 100 μL. Mix thoroughly with a pipette and spread the culture onto a solid LB culture dish containing Rif and Carb. Incubate in a 28°C incubator for 48 hours. After plaques develop, select suitable single colonies in the sterile environment of a clean bench. After successful transformation, confirm positive results from PCR testing and expand the culture. Aliquot the culture into 1.5 mL centrifuge tubes, add 60% glycerol, and store at -80°C for subsequent tobacco transfection.

[0095] (2) Injection of transfected tobacco and sampling

[0096] ①Solution configuration:

[0097] LB medium (1 L): 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and ddH2O to make up the volume.

[0098] 0.1M MES (pH 5.6) (1 L): 19.524 g MES, adjust the pH to 5.6 with NaOH, make up to volume with ddH2O, filter sterilize with a 0.22 μm filter, and store at room temperature.

[0099] 100 mM acetosyringone (AS): 0.036 g acetosyringone was dissolved in 1 mL dimethyl sulfoxide (DMSO).

[0100] 1M MgCl2 (400mL): 95.2g MgCl2·H2O, make up to volume with ddH2O, and store at room temperature.

[0101] Transfection solution: 10 mM MES, 100 μM AS, 10 mM MgCl2, and ddH2O to make up the volume.

[0102] ②Injection of tobacco

[0103] Agrobacterium containing the target plasmid was cultured under shaking conditions. About 5 mL of culture medium was collected and the pellet was collected by gentle centrifugation (room temperature, 6000 rpm, 5 minutes). The bacterial pellet was resuspended in the transfection solution and activated at 28°C, shaking at 100 rpm for 1 hour. Adjust the OD value of the activated culture to 0.1 for all three cultures. Transfect leaves were injected with a sterile syringe. Over the next two weeks, photograph and sample the transfected tobacco leaves.

[0104] (3) Western blot analysis

[0105] On the 7th day after transfection, 0.2 g of tobacco leaves expressing the gene were placed in a 1.5 mL centrifuge tube, quickly frozen in liquid nitrogen, and ground into powder using a grinding rod. 500 μL of NP-40 lysis buffer was added to the centrifuge tube, and the leaf powder was resuspended. The leaves were placed on ice for 10 min, and then centrifuged at 4°C, 14,000 rpm for 15 min. The supernatant was collected for Western blot analysis. Figure 6 The results showed that the expression levels were C3S5 > C3S2 > C3S1 > C3S3 > C3S4, and the positions of the bands from C3S1 to C3S5 were 46.3 kDa, 15.2 kDa, 35.8 kDa, 25.8 kDa, and 68.1 kDa, respectively.

[0106] (4) Real-time quantitative PCR (qRT-PCR) analysis

[0107] Primers were designed using Primer 5 software (Table 6) and synthesized by Qingke Biotechnology. RNA from five genes was extracted using the Omega R6827 kit. Reverse transcription (Vazyme kit) was used to generate cDNA as a template. Three biological replicates were performed using the real-time fluorescence quantitative PCR system (Table 7). Samples were applied to the membrane and centrifuged for mixing before qRT-PCR amplification.

[0108] like Figure 7 As shown, compared to unexpressed recombinant collagen, all five recombinant collagens were stably expressed in tobacco, with the expression levels ranking from highest to lowest in the order C3S5 > C3S2 > C3S1 > C3S3 > C3S4, consistent with Western blot analysis. Because C2S5 and C2S2 had high and similar expression levels, C3S5 and C3S2 were selected for subsequent experiments.

[0109] Table 6 qRT primer sequences

[0110] Primer name Sequence 5'→3' F-qRT-C3S1 ggtccatctggtcctgctgg R-qRT-C3S1 tcacctggcaaaccgttctc F-qRT-C3S2 ggtcctaagggagataaaggtgaacc R-qRT-C3S2 ctggagaaccatctctaccaggcaa F-qRT-C3S3 ttgagaggtggtgctggagag R-qRT-C3S3 ggtatgaagggtcatagaggttttga F-qRT-C3S4 caccagcgtgaccttgagg R-qRT-C3S4 taaggtggaccaataggacctggatga F-qRT-C3S5 gtgctccaggtcctaagggag R-qRT-C3S5 accaggtggacctataggaccag

[0111] Table 7 qPCR detection reaction system

[0112]

[0113] Example 3 Purification experiment of recombinant proteins C3S5 and C3S2

[0114] (1) Solution preparation

[0115] Plant cell lysate: 20 mM Tris-HCl, 4.8 mM beta-mercaptoethanol (β-ME), 1 mM phenylmethylsulfonyl fluoride (PMSF);

[0116] Equilibration solution: 20 mM Tris-HCl (pH 8.0);

[0117] Nonspecific protein elution buffer WB: 50 mM NaCl, 20 mM Tris-HCl (pH 8.0);

[0118] Protein elution buffer EB: 300 mM NaCl, 20 mM Tris-HCl (pH 8.0);

[0119] Dialysate: 20 mM Tris-HCl (pH 8.0);

[0120] Anion exchange chromatography filler: Cytiva Q Sepharose Fast Flow filler was used, the equilibration buffer was 20 mM Tris-HCl (pH 8.0), and the elution gradient was 0-300 mM NaCl;

[0121] Cation exchange chromatography filler: Cytiva SP Sepharose Fast Flow filler was used, the equilibration buffer was 20 mM sodium acetate (pH 4.5), and the elution gradient was 0-500 mM NaCl.

[0122] (2) Crude extract extraction

[0123] Tobacco leaves showing a clear phenotype after Agrobacterium transfection were removed, and the main veins of the leaves were removed. Weigh 1 kg of tobacco leaves containing C3S5 collagen fragments and place them in a chilled extraction buffer consisting of 20 mM Tris-HCl, 4.8 mM beta-mercaptoethanol (β-ME), and 1 mM phenylmethylsulfonyl fluoride (PMSF). 15 g of polyvinylpyridone (PVPP) and 6 g of activated carbon were added. Mixing was repeated five times, each lasting 1 minute, while maintaining the temperature below 15°C. The crude extract was filtered through a gauze pad and centrifuged for 30 minutes at 26,000 g at 5°C. The supernatant was collected and CaCl2 was added to a final concentration of 10 mM and activated carbon to a concentration of 1 g / L. Ficin (Sigma #F4125) was added at 5 mg / L; stirred for 3 hours at 15°C. Insoluble contaminants were removed by centrifugation (30 minutes at 22,000 g at 15°C). The C3S5 collagen fragment in the recovered supernatant was gradually precipitated by the addition of crystalline NaCl to a final concentration of 3.13 M (25 minutes at room temperature with continuous stirring). The solution was incubated in a refrigerator for 8 hours and then centrifuged (26,000 g for 2 hours at 5°C) to collect the pellets containing the C3S5 collagen fragment. The extraction method for the C3S2 collagen fragment pellets was the same as above.

[0124] (3) Purification of C3S5 and C3S5 fragment collagen

[0125] ②Cation exchange chromatography:

[0126] Separation and purification are achieved by utilizing the charge interaction between C3S5 collagen fragments and cation exchange media at low temperatures. 15 mL of SPFF filler is placed in a 50 mL gravity column, allowing the medium to settle freely and remaining in the column. The storage liquid is then drained, and the filler is compressed with a 20-micron circular sieve plate to stabilize it. 100 mL of equilibration solution is added to the column and allowed to flow through. Note that the filler must not dry out, so a small amount of liquid must remain on the upper sieve plate to complete the equilibration. After centrifugation of approximately 50 mL of the crude C3S5 collagen fragment extract, the supernatant is filtered through a 0.22-micron filter membrane to obtain the loading sample, which is then slowly added to the column. Gravity is used to allow the liquid to flow out, completing the loading.

[0127] ③ Rinse:

[0128] Use 100 mL of buffer solution, i.e., dialysate, to elute the gravity column, and then rinse the filler with 100 mL of non-specific protein eluent WB.

[0129] ⑤ Elution:

[0130] Use 50 mL of specific protein eluent EB to add the gravity column to elute the C3S5 fragment collagen, and collect the eluate in a 50 mL centrifuge tube.

[0131] Anion exchange chromatography:

[0132] Separation and purification are achieved by utilizing the charge interaction between C3S5 collagen fragments and anion exchange media at low temperatures. An appropriate amount of QSFF filler is placed in a gravity column, allowing the medium to settle freely and remaining in the column while the storage solution is drained. The filler is then compressed with a 20-micron circular sieve plate to stabilize it. 100 mL of equilibration solution is added to the column and allowed to flow through. Note that the filler must not dry out, so a small amount of liquid must remain on the upper sieve plate to complete the equilibrium. Approximately 50 mL of the eluate from the cationic chromatography step is filtered through a 0.22-micron filter membrane and then added directly to an anionic chromatography column. The flow-through is collected in a centrifuge tube.

[0133] ⑥ Dialysis:

[0134] Cut a dialysis bag of appropriate length with a molecular weight cutoff of 10 kDa. After cleaning and leak testing the dialysis bag, place the flow-through collected from the anions into 500 mL of pre-cooled dialysate and dialyze overnight in a 4°C refrigerator. Replace the dialysate every 12 hours for two changes.

[0135] ⑦Concentration:

[0136] Use a 50 mL 5 kDa ultrafiltration tube. Pre-chill the tube on ice for a few minutes and add 10-12 mL of the dialyzed sample to the upper portion of the tube. Balance the tube to achieve equilibrium in both mass and center of gravity. Centrifuge at 5000 × g for 15 minutes at 4°C in a high-speed refrigerated centrifuge. Repeat the centrifugation 2-4 times until the purified C3S5 collagen fragment sample is concentrated to approximately 1 mL.

[0137] The purification method of C3S2 fragment collagen sheet particles is the same as above.

[0138] Example 4: Functional detection of recombinant proteins C2S2 and C2S5

[0139] (1) Antioxidant test

[0140] According to Xin Xuanying's method (Xin Xuanying, Li Meiyao et al. 2023), the antioxidant activity of collagen was determined using vitamin C and butylated hydroxyanisole as the control group, and its in vitro antioxidant effect was explored by measuring the DPPH free radical and hydroxyl free radical scavenging ability.

[0141] DPPH free radical: Take five colorimetric tubes and add 3 mL of 0.05 mmol / L DPPH solution. Then, add 3 mL of collagen solution at 0.5, 1.0, 1.5, 2.0, or 3.0 mg / mL, respectively. Shake thoroughly, let stand for 30 minutes, and measure the absorbance (A1) at a wavelength of 517 nm. Add 3 mL of anhydrous ethanol to the gradient solution as the control group (A2). Mix 3 mL of anhydrous ethanol with 3 mL of DPPH solution as the blank group (A0). The DPPH free radical scavenging rate is calculated as follows: X1 = (1-(A1-A2) / A0) × 100%.

[0142] Hydroxyl free radicals: Mix 1 mL of 0.025 mol / L phosphate buffer (pH 7.4), 1 mL of 40 μg / mL safranin solution, 0.5 mL of 0.5, 1.0, 1.5, 2.0, and 3.0 mg / mL collagen solutions, 1 mL of 0.945 mmol / L sodium ferric EDTA, and 1 mL of 3% hydrogen peroxide solution in a 37°C water bath and heat for 30 min. The absorbance A1 was measured at a wavelength of 520 nm. The blank group A0 was treated with the addition of 0.5 mL of distilled water, and the control group was treated with the addition of 1.5 mL of distilled water. The hydroxyl free radical scavenging rate was calculated as follows: X2 = (A1-A0) / (A2-A0) × 100%.

[0143] The results showed that within the range of 0.5-3.0 mg / mL, higher concentrations of collagen solution had stronger antioxidant effects. C3S5 had scavenging rates of 82.5% and 42.3% for DPPH and hydroxyl radicals, respectively; while C3S2 had scavenging rates of 80.4% and 41.2% for DPPH and hydroxyl radicals, respectively.

[0144] (2) Cell adhesion assay

[0145] According to the method of Kang (Kang, Wang et al. 2018), with modifications, a 0.5 mg / mL recombinant collagen solution was added to a 96-well cell culture plate. A control group consisted of a heat-denatured 10% bovine serum albumin (BSA) solution and collagen standard. After incubation at 4°C for 24 hours, the supernatant was discarded. 100 μL of L929 suspension was added, and the cells were incubated at 37°C for 6–9 hours. Adherent cells were quantified by total DNA lysis, and three freeze-thaw cycles were performed with ultrapure water to lyse them. 5 μg / mL Hochest 33258 was added to the resulting cell lysate, and the cells were incubated in the dark for 1 hour. Absorbance was read using a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Each sample was assayed in triplicate, and data are expressed as mean ± standard deviation (SD).

[0146] like Figure 8The results show that the adhesion percentages of C3S2 were 120.42% and C3S5 were 130.43%, both higher than those of the collagen standard and BSA protein. This suggests that C3S2 and C3S5 are more readily absorbed and utilized by L929 cells than the collagen standard. The addition of C3S2 and C3S5 significantly promotes L929 cell adhesion and specific binding with adjacent cells or the extracellular matrix, facilitating cell regulation and potentially enabling applications in skin fibroblast repair.

[0147] (3) Cytotoxicity assay

[0148] According to the method of He Huixia (He Huixia, 2023), the toxicological effects of different concentrations of collagen solution on L929 cells were systematically evaluated by Cell Counting Kit-8 (CKK-8) to clarify its biocompatibility. Recombinant collagen solution samples with concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.3 mg / mL were prepared and filtered using a 0.22 μm filter membrane and stored at 4°C. 100 μL of L929 suspension (5×10 6 After culturing for 24 hours in a CO2 incubator at 37°C and 5% CO2, the culture medium was discarded. 100 μL of recombinant collagen solution of different concentrations was added. The DMEM culture medium with 100 μL was set as the control group. After culturing for 24 hours, the supernatant was discarded. 100 μL of 10% CKK-8 cell culture medium was added to each sample well and control well. After re-placement of the cell incubator and incubation for 2 hours, the absorbance at 450 nm was measured using an enzyme marker. Each sample was measured in parallel 3 times. The absorbance value of the sample group was A t The absorbance value of the control group was A c The absorbance value of the blank group is A0. The cell survival rate calculation formula (A t -A0) / (A c -A0)×100%.

[0149] The results showed that within the concentration range (0.01-0.3 mg / L), the cell activity of C3S2 and C3S5 gradually increased with the increase of concentration (e.g. Figure 9 ), among which C3S2 activity reached up to 120.3% and C3S5 activity reached up to 135.5%, indicating that these two proteins can effectively enhance the activity of organisms and do not cause toxicity to L929 cells.

[0150] (4) Biological scanning electron microscopy (SEM)

[0151] According to the method of Marin (Marin, Albu Kaya et al. 2018), recombinant collagen (50 μL, 1 mg / mL) was mixed with 5 μL of fibrogenesis buffer (200 mM Na₂HPO₄, pH 11.2) and incubated at 37°C for 1 h to induce fibrogenesis. Fibrils were collected by centrifugation (5 min, 13,000 rpm). Collagen fibril samples were immersed in 0.1 M phosphate buffer (pH 7.2) and 2.5% glutaraldehyde, rinsed five times in phosphate buffer, and then commissioned for testing by the Scientific Compass Testing Platform (Changsha).

[0152] The results show that at 1 μm, C3S2 and C3S5 are fibrous (e.g. Figure 10 ), showing its important role in biomechanics, cell interactions and tissue repair.

[0153] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A recombinant type III collagen fragment, characterized in that: Selected from: a) C3S1 protein having the amino acid sequence shown in SEQ 1; b) C3S2 protein having the amino acid sequence shown in SEQ2; c) C3S3 protein having the amino acid sequence shown in SEQ 3; d) C3S4 protein having the amino acid sequence shown in SEQ 4; e) C3S5 protein with the amino acid sequence shown in SEQ 5.

2. A tobacco transient expression system for efficient expression of recombinant proteins, characterized in that: Contains the plant expression vector pFolia40108, which uses the strong 35S promoter and NOS terminator.

3. A method for constructing a tobacco transient expression vector for collagen fragments, characterized in that: The steps are as follows: A1: Synthetic gene fragments C3S1, C3S2, C3S3, C3S4, and C3S5; A2: PCR amplification using primers F-C3S1 / R-C3S1, F-C3S2 / R-C3S2, F-C3S3 / R-C3S3, F-C3S4 / R-C3S4, and F-C3S5 / R-C3S5; A3: Add the amplified C3S1-C3S5 genes to the enzyme digestion system and double-digest with XhoI and BamHI; A4: Run the digestion product on agarose gel electrophoresis 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 enzymes, and ligated with T4 DNA ligase to obtain recombinant plasmids. A6 The recombinant plasmid was transformed into E. coli DH5α competent cells and screened on LB plates containing kanamycin; A7: Pick a single colony for amplification, extract the recombinant plasmid and verify its correctness by sequencing.

4. A method for transient expression of recombinant proteins C3S1, C3S2, C3S3, C3S4, and C3S5, characterized in that: The steps are as follows: B1: transforming the recombinant plasmid prepared according to claim 3 into Agrobacterium tumefaciens C58C1 strain by heat shock; B2: Use a sterile syringe to inject the bacterial solution into tobacco leaves for transfection; B3: Quantitative analysis by Western blot: the expression levels from high to low are C3S5>C3S2>C3S1>C3S3>C3S4.

5. The method according to claim 4, wherein The method for purifying C3S2 and C3S5 fragment collagen comprises: C1: Cation exchange chromatography: using Cytiva SP Sepharose Fast Flow filler, equilibration buffer: 20 mM sodium acetate (pH 4.5), elution gradient: 0-500 mM NaCl; C2: Anion exchange chromatography: using Cytiva Q Sepharose Fast Flow filler, equilibration buffer was 20 mM Tris-HCl (pH 8.0), and elution gradient was 0-300 mM NaCl; C3: Concentration: Place the eluate in a 10k (1 ± 20%) Da ultrafiltration centrifuge tube and centrifuge at 5000 (1 ± 20%) × g for 15 (1 ± 20%) minutes at 4 (1 ± 20%) °C. The final protein concentration is in the range of 5.3 to 7.4 mg / mL.

6. Use of C3S2 and C3S5 recombinant proteins in promoting cell adhesion.

7. Application of C3S2 and C3S5 recombinant proteins in antioxidant preparations.

8. Application of C3S2 and C3S5 recombinant proteins in cell repair.

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