Multifunctional i / iii chimeric recombinant humanized collagen fusion protein, coding gene and tissue repair application

Abstract

The application discloses a multifunctional I / III chimeric recombinant humanized collagen fusion protein, a coding gene and tissue repair application. The fusion protein comprises, from N terminal to C terminal, a human type I collagen core cell adhesion functional region, a first flexible connection peptide, a human type III collagen core proliferation promotion migration functional region, a second flexible connection peptide, an antibacterial and anti-inflammatory active polypeptide KRIVQRIKDFLRNLVPRTES and a C terminal three-helix stable repeat sequence. The application further provides a nucleic acid molecule coding the fusion protein, a recombinant expression vector pPIC9K-COl I / III containing the nucleic acid, a host cell Pichia pastoris GS115 engineering strain, and a method for preparing the fusion protein through high-density fermentation and four-step chromatography purification. The fusion protein has the functions of promoting cell adhesion, promoting cell proliferation, promoting cell migration, promoting angiogenesis, antibacterial and anti-inflammatory activity and excellent thermal stability (Tm is about 50.5 DEG C), and can be widely applied to the preparation of tissue repair materials, wound dressings, skin repair products and medical devices.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology, genetic engineering, protein engineering and biomedical materials technology, specifically relating to a multifunctional I / III chimeric recombinant humanized collagen fusion protein, a nucleic acid molecule encoding the fusion protein, an expression vector and host cell containing the nucleic acid molecule, a method for preparing the fusion protein, and its application in tissue repair materials, wound dressings, skin repair products and medical devices. Background Technology

[0002] Collagen is the most abundant structural protein in mammals. As a major component of the extracellular matrix, it plays an irreplaceable role in maintaining tissue integrity, regulating cell behavior, and participating in tissue repair and regeneration. More than 28 types of collagen are known to exist in the human body. Type I collagen is mainly found in the dermis of the skin, bones, and tendons, providing tensile strength to tissues. Type III collagen is mainly distributed in elastic tissues such as blood vessel walls, intestines, and skin; it is synthesized first during early wound healing and is subsequently gradually replaced by type I collagen.

[0003] Traditional sources of collagen mainly rely on extraction from animal tissues such as pig skin and bovine Achilles tendon. However, animal-derived collagen has significant limitations in clinical applications: first, there is the potential immunogenicity issue, as collagen from foreign sources can easily trigger an immune rejection response in the body; second, there is a risk of transmitting animal-derived viruses; and third, there are quality variations between different batches of animal-derived materials, making standardized production difficult.

[0004] With the development of genetic engineering and synthetic biology technologies, recombinant collagen technology has emerged. Recombinant collagen is produced by introducing the gene sequence of human collagen into engineered bacteria for expression. Its amino acid sequence is the same as or highly similar to human collagen, exhibiting low immunogenicity, good biocompatibility, and bioactivity. For example, Chinese patent CN118955688A discloses a recombinant fragmented humanized type III collagen, whose amino acid sequence is shown in SEQ ID NO.3. However, this type of technology only involves a single type of collagen fragment, resulting in a relatively limited function. Chinese patent CN118772263A discloses a recombinant human type III collagen that can promote cell migration; similarly, it belongs to a single type of recombinant collagen and has significant limitations in terms of functional diversity.

[0005] To further enhance the function of recombinant collagen, existing technologies attempt to combine type I and type III collagen. Chinese patent CN117285616A discloses a recombinant humanized type I+III collagen, composed of truncated human type I and type III collagen, exhibiting cell proliferation promotion and good water solubility. Chinese patent CN121135897B discloses a recombinant type I & III fusion collagen, and Chinese patent CN121135898B discloses a highly stable, low-immunogenic recombinant type I & III fusion collagen. However, most of these existing recombinant collagens are fragmented designs, lacking a complete triple-helix structure or exhibiting insufficient stability of the triple-helix structure, leading to decreased resistance to proteases. More importantly, none of these technologies involve anti-infection function design, requiring additional antibiotics for infected wounds in clinical applications, increasing the risk of drug resistance and treatment complexity.

[0006] In addition, non-patent literature Ma et al. (Bioresources and Bioprocessing, 2022) reported the expression of recombinant human collagen-like protein (RHLC) designed based on hydrophilic amino acids of type I and type III human collagen in Pichia pastoris GS115, with an expression level of 2.33 g / L in a 5-L fermenter, exhibiting a triple helix structure. Wang et al. (Curr Protoc., 2025) reported the expression of transdermal peptide-type III collagen fusion protein in Pichia pastoris GS115, with a yield of 1.9 g / L in a 15-L fermenter.

[0007] However, none of the above studies fused antimicrobial peptides with collagen at the gene level, nor did they introduce a triple-helix stabilizing sequence at the C-terminus to systematically improve thermal stability.

[0008] Regarding the application of antimicrobial peptides, Chinese patent CN116396403B discloses a recombinant fibronectin functional short form that fuses snake venom antimicrobial peptides with fibronectin fragments for expression. However, its protein backbone is not type I / III collagen, and the source and sequence of the antimicrobial peptides are completely different from those of this invention. In the prior art, the binding of antimicrobial peptides to collagen is mostly through physical blending, such as loading antimicrobial peptides into collagen-based hydrogels. However, physical blending suffers from problems such as burst release of antimicrobial peptides and difficulty in maintaining their activity.

[0009] Therefore, there is an urgent need in the field for a multifunctional recombinant collagen fusion protein that combines structural support, cell activity regulation, and active anti-infection capabilities. This protein should also have good thermal stability and triple helix structural integrity to meet the clinical needs of complex tissue repair, especially the repair of infected wounds. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multifunctional I / III chimeric recombinant humanized collagen fusion protein. Through rational design, the functional regions of human type I collagen and type III collagen are chimeric and fused with bioactive peptides with antibacterial and anti-inflammatory functions, and a triple-helix stable repeat sequence is introduced at the C-terminus to obtain a multifunctional fusion protein that simultaneously promotes cell adhesion, cell proliferation, cell migration, angiogenesis, antibacterial and anti-inflammatory effects, and good thermal stability.

[0011] Another object of the present invention is to provide a nucleic acid molecule encoding the fusion protein, a recombinant expression vector containing the nucleic acid molecule, and a host cell.

[0012] Another object of the present invention is to provide a method for preparing the fusion protein, including a high-density fermentation process and a separation and purification process.

[0013] Another object of the present invention is to provide the application of the fusion protein in the preparation of tissue repair materials, wound dressings, skin repair products and medical devices, particularly for the repair of infected wounds.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] A multifunctional I / III chimeric recombinant humanized collagen fusion protein, comprising, from the N-terminus to the C-terminus,: (i) a human type I collagen core cell adhesion functional region, the amino acid sequence of which is shown in SEQ ID NO:1; (ii) a first flexible linker peptide; (iii) a human type III collagen core proliferation and migration functional region, the amino acid sequence of which is shown in SEQ ID NO:2; (iv) a second flexible linker peptide; (v) an antibacterial and anti-inflammatory active polypeptide, the amino acid sequence of which is shown in SEQ ID NO:3; and (vi) a C-terminal triple-helix stable repeat sequence, the amino acid sequence of which is shown in SEQ ID NO:4; wherein the fusion protein possesses cell adhesion-promoting, cell proliferation-promoting, cell migration-promoting, angiogenesis-promoting, antibacterial and anti-inflammatory activities, and thermal stability.

[0016] The amino acid sequence of the first flexible linker peptide is GGGGSGGGGS, and the amino acid sequence of the second flexible linker peptide is GGGGS.

[0017] The mature amino acid sequence of the fusion protein is shown in SEQ ID NO:5.

[0018] The fusion protein, when expressed, further includes an N-terminal signal peptide, which is the Pichia pastoris α-mating factor secretion signal peptide, the amino acid sequence of which is shown in SEQ ID NO:6; the complete precursor amino acid sequence containing this signal peptide is shown in SEQ ID NO:7.

[0019] The present invention also provides a nucleic acid molecule encoding the above-mentioned fusion protein. Preferably, the nucleotide sequence of the nucleic acid molecule is an optimized sequence based on the codon bias of the Pichia pastoris expression system.

[0020] The present invention also provides a recombinant expression vector containing the above-mentioned nucleic acid molecule. Preferably, the recombinant expression vector is the Pichia pastoris secretory expression vector pPIC9K digested with EcoRI and NotI, so that the nucleic acid molecule is located downstream of the α-mating factor secretion signal peptide, thereby constructing the recombinant expression vector pPIC9K-COlⅠ / Ⅲ.

[0021] The present invention also provides a host cell containing the above-described recombinant expression vector. Preferably, the host cell is an engineered strain of Pichia pastoris GS115.

[0022] The present invention also provides a method for preparing the above-mentioned fusion protein, comprising the following steps:

[0023] (1) Gene synthesis and construction of expression vector: The gene encoding the fusion protein (after codon optimization by Pichia pastoris) was synthesized, digested with EcoRI and NotI, and then ligated into the Pichia pastoris secretory expression vector pPIC9K which was also digested with the same enzymes, so that the target gene is located downstream of the α-mating factor secretion signal peptide, and the recombinant expression vector pPIC9K-COlⅠ / Ⅲ was constructed.

[0024] (2) Construction and screening of engineered bacteria: ① The recombinant expression vector pPIC9K-COlⅠ / Ⅲ was linearized with restriction endonuclease SalI and then transformed into Pichia pastoris GS115 competent cells by electroporation. The transformation product was plated on MD plates (without histidine) and cultured at 30℃ for 2-3 days to screen for histidine-deficient transformants. ② Transformants grown on MD plates were picked and sequentially inoculated onto YPD plates containing different concentrations of G418 (0.25, 0.5, 1.0, 2.0, 4.0 mg / mL) and cultured at 30℃ for 2-3 days to screen for high-copy-integration transformants. ③ The high-copy transformants obtained by screening were inoculated into YPD medium and cultured at 30℃ and 220 rpm with shaking until OD600≈2-6. The cells were collected by centrifugation, resuspended in BMMY medium for induction of expression, and methanol was added every 24 h to a final concentration of 0.5%. The cells were then induced and cultured at 28℃ for 72-96 h. The induction supernatant was subjected to SDS-PAGE and Western blot analysis to screen for the engineered strain with the highest expression level.

[0025] (3) High-density fermentation: ① Seed culture preparation: The engineered bacteria were inoculated into YPD liquid medium and cultured at 30℃ and 250 rpm for 18-24 h with shaking to obtain the primary seed culture. 5% of this primary seed culture was then transferred to BMGY medium and cultured at 30℃ and 250 rpm for 24 h with shaking to obtain the secondary seed culture. ② Fermentation tank culture: The secondary seed culture was inoculated into a fermenter containing basal salt medium (BSM) at a 10% inoculation rate. The fermentation conditions were: temperature 30℃, pH 5.0 (automatically adjusted with 25% ammonia water), and dissolved oxygen maintained at ≥20%. The initial glycerol fermentation stage lasted approximately 18-24 hours. When dissolved oxygen was >80%, 50% glycerol (containing 12 mL / L PTM1 trace elements) was added for fed-batch glycerol culture for approximately 4-6 hours. When the cell wet weight reached 180-220 g / L, the addition of glycerol was stopped. When the dissolved oxygen rose above 70%, the cells were allowed to stand for 30 minutes before entering the methanol induction stage. ③ Methanol induction stage: 100% methanol (containing 12 mL / L PTM1 trace elements) was added to induce expression. The methanol flow rate was controlled at 12 g / L, dissolved oxygen was maintained above 20%, and the induction temperature was adjusted to 28℃. The induction time lasted for 72-96 hours. The induction expression was stopped when the cell wet weight no longer increased. During the fermentation process, samples were taken every 24 hours to detect cell wet weight and protein expression levels.

[0026] (4) Separation and purification: ① Fermentation broth pretreatment: After fermentation, the fermentation broth was centrifuged at 10,000 rpm for 10 min at 4℃ to remove the bacterial cells and obtain the supernatant. ② Microfiltration was performed using a tangential flow microfiltration membrane to remove bacterial fragments and large protein aggregates. ③ Dialysis was performed using a dialysis bag with 20 mM Tris-HCl buffer for dialysis for 36 h. ④ Ion exchange chromatography: The dialyzed protein solution was loaded at a flow rate of 1 mL / min onto a HiTrap Q HP anion exchange chromatography column pre-equilibrated with binding buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, pH 8.0). A linear gradient elution was performed using Tris-HCl buffer (pH 8.0) containing 2 M NaCl, and the target protein peak was collected. ⑤ The protein solution collected by ion exchange chromatography was concentrated and loaded onto a Superdex 200 micrometer. Increase the gel filtration chromatography column, elute isocratically with PBS buffer (pH 7.4), and collect the target protein peak; ⑥ Concentrate the protein solution collected by gel filtration chromatography using an ultrafiltration tube (molecular weight cutoff 10 kDa), then filter it through a 0.22 μm filter membrane for sterilization, aliquot and freeze-dry to obtain fusion protein lyophilized powder.

[0027] This invention has the following outstanding advantages:

[0028] 1. Multifunctional Integration and Synergistic Effect: This invention systematically integrates and fuses the core cell adhesion functional region of human type I collagen, the cell proliferation and migration-promoting functional region of human type III collagen, and bioactive peptides with broad-spectrum antibacterial and anti-inflammatory activities through optimized flexible linker peptides, constructing a novel fusion protein with triple functions of structural support, cell activity regulation, and anti-infection. Each functional module maintains relatively independent and correct folding through the flexible linker peptides, while the overall structural stability is enhanced by the C-terminal triple helix stabilizing sequence, resulting in a significant synergistic effect.

[0029] 2. Active anti-infection capability: The antibacterial and anti-inflammatory functional modules in the fusion protein endow the product with the ability to actively resist pathogenic microorganisms and regulate inflammatory responses. It can effectively improve the microenvironment for the repair of infected wounds without the need for additional antibiotics, and reduce the risk of drug resistance.

[0030] 3. Excellent thermal stability and resistance to degradation: By designing a stable triple helix repeating sequence at the C-terminus and combining it with the natural triple helix forming ability of the functional region of type I / III collagen, the fusion protein of this invention has a higher thermal denaturation temperature (Tm of about 50.5°C, which is about 10.5°C higher than the control group) and stronger resistance to protease degradation, thus prolonging the effective duration of action in vivo.

[0031] 4. Angiogenesis activity: The type I collagen functional region of the fusion protein contains multiple integrin binding sites, which can effectively promote the adhesion, proliferation and migration of vascular endothelial cells, thereby promoting wound angiogenesis.

[0032] 5. Low immunogenicity: All functional modules are derived from human natural sequences or human protein derivative sequences, are highly homologous to human proteins, have low immunogenicity, and excellent biocompatibility.

[0033] 6. High expression and purification efficiency: This invention optimizes the coding gene sequence of the fusion protein, which is suitable for the codon bias of the Pichia pastoris expression system, and achieves high-level secretory expression. Through a combined purification strategy of tangential flow microfiltration-anion exchange-gel filtration, the target protein with a purity of ≥95% can be obtained, which is suitable for large-scale industrial production. Attached Figure Description

[0034] Figure 1 This is the SDS-PAGE electrophoresis result of the recombinant fusion protein. The marker is the protein molecular weight standard, and 1 / 2 / 3 are purified samples from three high-copy engineered bacteria.

[0035] Figure 2 The image shows the results of circular dichroism (CD) analysis of the recombinant fusion protein, which displays a typical positive peak at 220 nm and a secondary negative peak at 208 nm, confirming the triple helix conformation.

[0036] Figure 3 The image shows the results of the cell adhesion experiment, indicating that the adhesion rate of the fusion protein was significantly higher than that of the control group.

[0037] Figure 4 The image shows the results of the cell migration experiment (scratch healing experiment), which shows that the cell migration rate of the fusion protein group was significantly higher than that of the single protein group and the blank group after 24 hours.

[0038] Figure 5 The image shows the healing effect of infected wounds in rats, indicating that the wound closure rate was highest in the fusion protein group on day 14. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below through specific embodiments. These embodiments are merely illustrative and are not intended to limit the scope of protection of the present invention.

[0040] Example 1: Gene design of recombinant fusion protein

[0041] The mature peptide of the multifunctional type I / III chimeric recombinant humanized collagen fusion protein of the present invention is composed of the following components from the N-terminus to the C-terminus:

[0042] 1. Core cell adhesion functional region of human type I collagen (SEQ ID NO: 1): Arg-Gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg-Gly-Pro-Ala-Gly-Pro-Gln-Gly-Pro-Arg-Gly-Asp-Lys-Gly-Glu-Thr-Gly-Glu-Gln-Gly-Asp-Arg-Gly-Ile-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Leu-Gln-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Ser-Pro-Gly-Glu-Gln-Gly-Pro-Ser-Gly-Ala-Ser-Gly-Pro-Ala-Gly-Pro-Arg-Gly-Pro-Pro-Gly-Ser-Ala-Gly-Ala-Pro-Gly-Lys-Asp-Gly-Leu-Asn-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Leu-Pro-Gly-Met-Ala-Gly-Ser-Arg-Gly-Gly-Pro-Gly-Leu-Pro-Gly-Pro-Lys-Gly-Asp-Arg-Gly-Asp-Ala-Gly-Pro-Lys-Gly-Ala-Asp-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-Pro-Pro-Gly-Pro-Arg

[0043] 2. First flexible connecting peptide: GGGGSGGGGS

[0044] 3. Core proliferative and migration-promoting functional region of human type III collagen (SEQ ID NO:2): Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala-Pro-Gly-Glu-Arg-Gly-Pro-Pro-Gly-Pro-Gln-Gly-Pro-Arg-Gly-Asp-Lys-Gly-Glu-Thr-Gly-Glu-Gln-Gly-Asp-Arg-Gly-Ile-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Leu-Gln-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Ser-Pro-Gly-Glu-Gln-Gly-Pro-Ser-Gly-Ala-Ser-Gly-Pro-Ala-Gly-Pro-Arg-Gly-Pro-Pro-Gly-S er-Ala-Gly-Ala-Pro-Gly-Lys-Asp-Gly-Leu-Asn-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Leu-Pro-Gly-Met-Ala-Gly-Ser-Arg-Gly-Gly- Pro-Gly-Leu-Pro-Gly-Pro-Lys-Gly-Asp-Arg-Gly-Asp-Ala-Gly-Pro-Lys-Gly-Ala-Asp-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-Pro-Pro-Gly-Pro-Arg

[0045] 4. Second flexible linker peptide: GGGGS

[0046] 5. Antibacterial and anti-inflammatory active peptide (SEQ ID NO:3): Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser

[0047] 6. C-terminal triple-helix stable repeat sequence (SEQ ID NO:4): Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Leu

[0048] 7. The complete amino acid sequence of the mature fusion protein (SEQ ID NO:5):

[0049]

[0050] 8. In the expression system, the N-terminus of the mature fusion protein is further fused with a Pichia pastoris α-mating factor secretion signal peptide (SEQ ID NO:6):

[0051] Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe-Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val- Asn-Thr-Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu-Ala-Val-Ile-Gly-Tyr-Ser-Asp-Leu-G lu-Gly-Asp-Phe-Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn-Asn-Gly-Leu-Leu-Phe-Ile-As n-Thr-Thr-Ile-Ala-Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala

[0052] 9. Used to guide the secretory expression of recombinant proteins. The complete amino acid sequence of the precursor protein containing this signal peptide (SEQ ID NO:7) is shown:

[0053]

[0054] 10. The nucleic acid molecule (SEQ ID NO:8) encoding the above-mentioned mature fusion protein was synthesized with optimized results based on the codon bias of Pichia pastoris:

[0055]

[0056] Example 2 Construction of recombinant expression vector and screening of engineered bacteria

[0057] The codon-optimized gene (SEQ ID NO:8) from Example 1 was synthesized by Sangon Biotech Co., Ltd. The synthesized gene was double-digested with EcoRI and NotI and then ligated into the Pichia pastoris secretory expression vector pPIC9K, which had also been double-digested, placing the target gene downstream of the α-mating factor secretion signal peptide (SEQ ID NO:6), thus constructing the recombinant expression vector pPIC9K-COlⅠ / Ⅲ.

[0058] The recombinant expression vector pPIC9K-COlⅠ / Ⅲ was linearized with the restriction endonuclease SalI and then transformed into Pichia pastoris GS115 competent cells by electroporation. The transformation product was plated on MD plates (histidine-free) and incubated at 30°C for 2-3 days to screen for histidine-deficient transformants.

[0059] Transformants grown on MD plates were picked and sequentially seeded onto YPD plates containing different concentrations of G418 (0.25, 0.5, 1.0, 2.0, 4.0 mg / mL). The plates were incubated at 30°C for 2-3 days to screen for high-copy-integration transformants.

[0060] The high-copy transformants obtained through screening were inoculated into YPD medium and cultured at 30℃ and 220 rpm with shaking until OD600≈2-6. The cells were collected by centrifugation and resuspended in BMMY medium for induction of expression. Methanol was added every 24 h to a final concentration of 0.5%, and induction was continued at 28℃ for 72-96 h. The induction supernatant was analyzed by SDS-PAGE and Western blot to screen for the engineered strain with the highest expression level, named GS115 / pPIC9K-COlⅠ / Ⅲ-High.

[0061] Example 3 High-density fermentation

[0062] The engineered strain GS115 / pPIC9K-COlⅠ / Ⅲ-High obtained in Example 2 was subjected to high-density fermentation:

[0063] (1) Seed culture preparation: The engineered bacteria were inoculated into YPD liquid medium and cultured at 30℃ and 250 rpm for 18-24 h with shaking to obtain the primary seed culture. The culture was then transferred to BMGY medium at an inoculation rate of 5% and cultured at 30℃ and 250 rpm for 24 h with shaking to obtain the secondary seed culture.

[0064] (2) Fermentation tank culture: The secondary seed culture was inoculated into a 5L fermenter containing basal salt medium (BSM) at a 10% inoculation rate. The fermentation conditions were: temperature 30℃, pH 5.0 (automatically adjusted with 25% ammonia water), and dissolved oxygen maintained at ≥20%. The initial glycerol fermentation stage was maintained for about 18-24 hours. When the dissolved oxygen was >80%, 50% glycerol (containing 12mL / L PTM1 trace element) was added for fed-batch glycerol culture for about 4-6 hours. When the cell wet weight reached 180-220g / L, the addition of glycerol was stopped. When the dissolved oxygen rose to above 70%, the culture was allowed to stand for 30 minutes to enter the methanol induction stage.

[0065] (3) Methanol induction stage: 100% methanol (containing 12 mL / L PTM1 trace elements) was added to induce expression. The methanol flow rate was controlled at 12 g / L, dissolved oxygen was maintained above 20%, the induction temperature was adjusted to 28℃, and the induction time lasted for 72-96 h. Induction was stopped when the wet weight of the cells stopped increasing. During the fermentation process, samples were taken every 24 h to detect the wet weight of the cells and the protein expression level.

[0066] Example 4 Separation and Purification

[0067] (1) Pretreatment of fermentation broth: After fermentation, the fermentation broth was centrifuged at 10,000 rpm for 10 min at 4℃ to remove the cells and obtain the supernatant.

[0068] (2) Tangential flow microfiltration: tangential flow microfiltration membrane is used to remove bacterial fragments and large molecular protein aggregates.

[0069] (3) Dialysis solution change: Dialyze the dialysis bag with 20mM Tris-HCl buffer for 36h.

[0070] (4) Anion exchange chromatography: The dialyzed protein solution was loaded onto a HiTrap Q HP anion exchange chromatography column pre-equilibrated with binding buffer (20mM Tris-HCl, 500mM NaCl, 1mM EDTA, pH 8.0) at a flow rate of 1mL / min. The target protein peak was collected by linear gradient elution with Tris-HCl buffer (pH 8.0) containing 2M NaCl.

[0071] (5) Gel filtration chromatography: After concentrating the protein solution collected by ion exchange chromatography, the protein solution was loaded onto a Superdex 200 Increase gel filtration chromatography column and eluted isocratically with PBS buffer (pH 7.4) to collect the target protein peak.

[0072] (6) Concentration and sterilization: The protein solution collected by gel filtration chromatography was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, then filtered and sterilized using a 0.22 μm filter membrane, and then freeze-dried after dispensing to obtain fusion protein lyophilized powder.

[0073] Example 5 Protein Identification and Structural Characterization

[0074] 1. SDS-PAGE electrophoresis identification

[0075] The lyophilized powder obtained in Example 4 was reconstituted and subjected to SDS-PAGE electrophoresis. The theoretical molecular weight of the fusion protein of this invention is approximately 41 kDa. Due to the special structure of collagen, which is rich in proline, its apparent molecular weight is 45-50 kDa. SDS-PAGE showed a single protein band of approximately 50 kDa with a purity ≥95% (see...). Figure 1 Note: 1, 2, and 3 represent different strains of the fusion protein.

[0076] 2. Circular dichroism (CD) spectroscopy analysis

[0077] The purified fusion protein was diluted to 0.2 mg / mL with PBS and subjected to CD spectral scanning. The results showed that the fusion protein exhibited a typical positive peak at 220 nm, consistent with the characteristic spectrum of a triple helix structure; a secondary negative peak was also observed at 208 nm, further confirming its ordered triple helix conformation (see...). Figure 2 ).

[0078] 3. Thermal stability analysis

[0079] Differential scanning calorimetry (DSC) was used with a heating rate of 1℃ / min and a temperature range of 20-80℃. The thermal denaturation process was monitored by the change in ellipticity at 220nm. The experimental results in Table 1 show that the thermal denaturation temperature (Tm) of the fusion protein of this invention is 50.5℃, which is approximately 10.5℃ higher than the control group of single-type recombinant collagen fragments (40.0℃), indicating that the fusion design significantly enhances the thermal stability of the protein.

[0080] Table 1. Experimental results of thermal stability analysis

[0081]

[0082] Example 6: Evaluation of in vitro biological activity

[0083] 1. Cell adhesion-promoting activity

[0084] The CCK-8 assay was used. The fusion protein was diluted with PBS to the specified concentration (1-200 μg / mL), coated with 96-well plates (overnight at 4°C), blocked with 1% BSA for 1 h, and then seeded with L929 fibroblasts. After culturing at 37°C and 5% CO2 for 4 h, CCK-8 reagent was added, and the cells were cultured for another 2 h. The absorbance at 450 nm was measured using a microplate reader.

[0085] The results showed that, compared with the control group, the adhesion rate of the fusion protein increased with increasing concentration at 30 min and 1 h, and the adhesion rate of the fusion protein was significantly higher than that of the control group (see...). Figure 3 ).

[0086] 2. Cell proliferation-promoting activity

[0087] The CCK-8 assay was used. L929 cells were seeded into 96-well plates and cultured for 24 h. The medium was then replaced with a medium containing different concentrations of fusion protein (1-500 μg / mL), and the cells were cultured for another 48 h. CCK-8 reagent was then added, and the absorbance at 450 nm was measured using a microplate reader.

[0088] The results are shown in Table 2. The effective concentration ranged from 1 to 100 μg / mL, with the optimal concentration being 25 μg / mL. At a concentration of 25 μg / mL, the cell proliferation rate of the fusion protein group reached 212.03%, which was approximately 70% higher than that of the control group with the single protein (141.72%).

[0089] Table 2 Analysis of cell proliferation-promoting activities

[0090]

[0091] 3. Promotes cell migration

[0092] Scratch healing assay was performed. L929 cells were cultured in 6-well plates until confluence. Vertical scratches were made using a 200 μL pipette tip. After washing with PBS, serum-free medium containing either the fusion protein (100 μg / mL) or the control group's single protein (100 μg / mL) was added. Images were taken under an inverted microscope at 0 h, 6 h, and 24 h, and the remaining scratch area was measured using ImageJ.

[0093] The results showed that at 24 hours, the cell migration rates were: 70% in the control group, 96.78% in the fusion protein group, and 90.84% ​​in the single protein group. The cell migration-promoting ability of the fusion protein was significantly higher than that of the single protein group (see...). Figure 4 ).

[0094] Example 7: In vivo tissue repair effect

[0095] To investigate the promoting effect of the fusion protein of this invention on the repair of infected wounds, a rat model of full-thickness skin infected wound on the back was established (inoculated with Staphylococcus aureus). The experiment was divided into three groups: blank group (physiological saline), single protein group (single recombinant type III collagen, 100 μg / mL), and fusion protein group (fusion protein of this invention, 100 μg / mL). The wound closure status was recorded by photography on days 0, 7, and 14.

[0096] The results showed that on day 7, the wound closure area was similar in the control group and the experimental group; on day 14, the wound closure rate of all groups was close to 90% or higher, with the fusion protein group showing the highest closure rate at 97.85%; the healing rate of the control group was 91.82%, and the healing rate of the single protein group was 93.11%. Histological analysis showed that the fusion protein group had thicker new epidermis, more regular collagen fiber arrangement, and less inflammatory cell infiltration. This indicates that the fusion protein of the present invention can significantly promote the closure of infected wounds and tissue regeneration (see...). Figure 5 ).

[0097] Example 8 Antibacterial activity assay

[0098] The minimum inhibitory concentration (MIC) of the fusion protein against Staphylococcus aureus and Escherichia coli was determined using the microbroth dilution method. The fusion protein was serially diluted with PBS, mixed with bacterial culture, and incubated at 37°C for 18 h before the OD600 was measured.

[0099] The results showed that the MIC of the fusion protein against Staphylococcus aureus was 16 μg / mL and the MIC against Escherichia coli was 32 μg / mL, indicating that the fusion protein retained the broad-spectrum antibacterial activity of the antibacterial and anti-inflammatory peptides.

Claims

1. A multifunctional I / III chimeric recombinant humanized collagen fusion protein, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, the following: (i) a human type I collagen core cell adhesion functional region, the amino acid sequence of which is shown in SEQ ID NO:1; (ii) a first flexible linker peptide; (iii) a human type III collagen core proliferation and migration functional region, the amino acid sequence of which is shown in SEQ ID NO:2; (iv) a second flexible linker peptide; (v) an antibacterial and anti-inflammatory active polypeptide, the amino acid sequence of which is shown in SEQ ID NO:3; and (vi) a C-terminal triple helix stable repeat sequence, the amino acid sequence of which is shown in SEQ ID NO:4; wherein, the fusion protein has cell adhesion-promoting, cell proliferation-promoting, cell migration-promoting, angiogenesis-promoting, antibacterial and anti-inflammatory activities and thermal stability.

2. The fusion protein according to claim 1, characterized in that, The first flexible linker peptide and the second flexible linker peptide are each independently selected from (GGGGS)n, where n is an integer from 1 to 3; the amino acid sequence of the first flexible linker peptide is GGGGSGGGGS, and the amino acid sequence of the second flexible linker peptide is GGGGS.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:5; or, the fusion protein further comprises an N-terminal signal peptide, which is the Pichia pastoris α-mating factor secretion signal peptide, the amino acid sequence of which is shown in SEQ ID NO:6, and the complete precursor amino acid sequence of the fusion protein is shown in SEQ ID NO:

7.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid molecule is an optimized sequence based on the codon bias of the Pichia pastoris expression system.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 4 or 5; the recombinant expression vector is the Pichia pastoris secretory expression vector pPIC9K, which is digested with EcoRI and NotI, so that the nucleic acid molecule is located downstream of the α-mating factor secretion signal peptide, and the recombinant expression vector pPIC9K-COlⅠ / Ⅲ is constructed.

7. A host cell, characterized in that, The host cell contains the recombinant expression vector as described in claim 6; the host cell is the Pichia pastoris GS115 engineered strain.

8. A method for preparing the fusion protein according to any one of claims 1-3, characterized in that, Includes the following steps: (a) Gene synthesis and expression vector construction: The gene encoding the fusion protein was synthesized, digested with EcoRI and NotI, and then ligated into the Pichia pastoris secretory expression vector pPIC9K, which was also digested with the same enzymes, so that the target gene was located downstream of the α-mating factor secretion signal peptide, and the recombinant expression vector pPIC9K-COlⅠ / Ⅲ was constructed. (b) Construction and screening of engineered strains: The recombinant expression vector was linearized with restriction endonuclease SalI and then transformed into Pichia pastoris GS115 competent cells by electroporation. Histidine-deficient transformants were screened by MD plates, and high-copy-count integrated transformants were screened by G418 resistance gradient. The engineered strains with the highest expression levels were further screened by methanol induction in BMMY medium and SDS-PAGE and Western blot analysis. (c) High-density fermentation: The selected engineered strains were cultured sequentially with YPD primary seed culture and BMGY secondary seed culture. They were then inoculated into a fermenter containing BSM basal salt medium at a 10% inoculum. The initial glycerol fermentation stage was maintained for 18-24 hours. When the dissolved oxygen level reached above 80%, 50% glycerol was fed in for glycerol fed-batch culture for 4-6 hours until the cell wet weight reached 180-220 g / L. The glycerol feeding was then stopped. After the dissolved oxygen level reached above 70% and the culture was allowed to stand for 30 minutes, the methanol induction stage was started. 100% methanol was fed in to induce expression. The methanol feed rate was controlled at 12 g / L, the dissolved oxygen level was maintained above 20%, the induction temperature was 28℃, and the induction time was 72-96 hours. The fermentation conditions were: temperature 30℃, pH 5.0 automatically regulated with 25% ammonia, and dissolved oxygen maintained at ≥20%; the 50% glycerol contained 12 mL / L of PTM1 trace element, and the 100% methanol contained 12 mL / L of PTM1 trace element. (d) Separation and purification: After fermentation, the supernatant was collected by centrifugation and then subjected to tangential flow microfiltration, dialyzing with 20mM Tris-HCl buffer for 36h, linear gradient elution by HiTrap Q HP anion exchange chromatography, isocratic elution by Superdex 200 Increase gel filtration chromatography, ultrafiltration concentration, and sterilization by filtration through a 0.22μm filter membrane. Finally, the fusion protein was freeze-dried to obtain lyophilized powder. The binding buffer for the anion exchange chromatography was 20 mM Tris-HCl, 500 mM NaCl, and 1 mM EDTA, pH 8.0; the elution buffer was Tris-HCl buffer containing 2 M NaCl, pH 8.0; the gel filtration chromatography was performed isocratic elution with PBS buffer, pH 7.4; and the ultrafiltration was performed using an ultrafiltration tube with a molecular weight cutoff of 10 kDa.

9. The use of the fusion protein according to any one of claims 1-3 in the preparation of tissue repair materials, wound dressings, skin repair products or medical devices.

10. The application according to claim 9, characterized in that, The tissue repair materials, wound dressings, skin repair products, or medical devices are used for the repair of infected wounds.

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