A nucleic acid molecule encoding human defensin and a method for preparing human defensin and its application

Abstract

The present invention belongs to the fields of genetic engineering, bioengineering and biomedicine, and relates to a nucleic acid molecule encoding human defensin and a method and application for preparing human defensin. The nucleic acid sequence of the nucleic acid molecule includes any one of the following: (1) a sequence as shown in SEQ ID NO.2; (2) a nucleic acid sequence encoding human defensin obtained by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO.2; (3) a nucleic acid sequence having at least 80% sequence homology with the nucleic acid sequence described in (1) or (2) and having the same or similar functions. The method provided by the present invention uses a nucleic acid molecule encoding human defensin to construct a recombinant expression vector, which can recombinantly express human defensin hBD‑3, release the recombinant fusion protein into the culture supernatant, and facilitate protein purification. The recombinant human defensin obtained by the present invention has tissue regeneration function, and provides a new active molecule for the development of new drugs.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering, bioengineering and biomedicine, and relates to a nucleic acid molecule encoding human defensin, a method for preparing human defensin and its application. Background Art

[0002] Human beta-defensin 3 (hBD-3) is a naturally occurring antimicrobial peptide in the human body, belonging to the beta-defensin family. This class of peptides primarily participates in the body's innate immunity, combating infection by directly killing microorganisms and modulating the immune system. hBD-3 exhibits broad-spectrum antimicrobial activity, effectively targeting not only bacteria (including Gram-positive and Gram-negative bacteria) and fungi, but also certain viruses (such as human immunodeficiency virus (HIV)). hBD-3 exerts its antimicrobial activity by disrupting pathogen cell membranes. It interacts with the anionic membrane of pathogens, leading to membrane disruption and pathogen death.

[0003] Due to its potent antimicrobial, immunomodulatory, and tissue regenerative properties, hBD-3 has been extensively studied for its applications in anti-infective therapy, wound healing, and oral mucosal and vaginal regeneration. Amid the growing threat of antimicrobial resistance, hBD-3 represents a promising alternative to antibiotics or adjunctive therapy. In summary, hBD-3, due to its potent antimicrobial and immunomodulatory properties, its unique advantages in promoting tissue regeneration and its low resistance-inducing potential, has become a key focus of future biomedical research and applications. However, human defensins are present in low biomass and difficult to extract, while chemical synthesis methods are costly. Therefore, genetic engineering is a cost-effective approach for their large-scale production. However, the complex eukaryotic protein hBD-3 is prone to misfolding and inclusion body formation in Escherichia coli, resulting in low refolding efficiency and low functional protein expression yield and activity. Consequently, the recombinant production of human defensins using prokaryotes is challenging, and ensuring the antimicrobial activity of the recombinant peptide is difficult, a key factor currently limiting the recombinant production of defensins.

[0004] Therefore, there is an urgent need to provide a method for effectively preparing recombinant defensins and new applications. Summary of the Invention

[0005] In response to the deficiencies in the prior art and actual needs, the present invention provides a nucleic acid molecule encoding human defensin, a method for preparing human defensin, and an application thereof. The present invention optimizes a recombinant expression vector of human defensin based on a pET-32a(+) vector framework, transforms the vector into an Escherichia coli competent Origami, and uses protein pilot tests and SDS-PAGE gel electrophoresis to explore expression conditions. It is found that human defensin and the pET-32a(+) recombinant expression vector successfully express a fusion protein Trx-hBD-3 in the supernatant. The fusion protein is cleaved by enterokinase, and the protein is purified according to the expressed protein tag to successfully obtain human defensin. The recombinant human defensin of the present invention has multiple functions such as antibacterial and promotion of collagen expression, and has potential application value.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a nucleic acid molecule encoding a human defensin, wherein the nucleic acid sequence of the nucleic acid molecule comprises any one of the following:

[0008] (1) the sequence shown in SEQ ID NO. 2;

[0009] (2) A nucleic acid sequence encoding a human defensin obtained by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO. 2;

[0010] (3) A nucleic acid sequence that has at least 80% sequence homology with the nucleic acid sequence described in (1) or (2) and has the same or similar functions.

[0011] The method of the present invention uses a nucleic acid molecule encoding human defensin to construct a recombinant expression vector, which can recombinantly express human defensin hBD-3 and release the recombinant fusion protein into the culture supernatant, facilitating protein purification. The recombinant human defensin obtained by the method of the present invention has tissue regeneration function and provides a new active molecule for the development of new drugs.

[0012] SEQ ID NO.2:

[0013] GGAATTATCAACACCCTGCAGAAATATTACTGCCGCGTGAGGGGAGGGAGATGTGCAGTGTTGAGTTGCTTACCAAAAGAAGAACAGATAGGGAAATGTAGTACCAGAGGAAGGAAATGTTGTAGAAGGAAGAAA.

[0014] In a second aspect, the present invention provides a vector comprising the nucleic acid molecule encoding the human defensin according to the first aspect.

[0015] In a third aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule encoding human defensin according to the first aspect or the vector according to the second aspect.

[0016] In a fourth aspect, the present invention provides a recombinant preparation method of human defensins, the recombinant preparation method comprising the following steps:

[0017] (1) constructing a recombinant plasmid containing the nucleic acid molecule encoding human defensin described in the first aspect, transforming it into competent cells, and culturing it to obtain a human defensin expression strain;

[0018] (2) Using inducers to induce the human defensin expression strain to express recombinant human defensin fusion protein;

[0019] (3) Purify the recombinant human defensin fusion protein obtained in step (2).

[0020] In the present invention, the nucleic acid molecule encoding human defensin can be obtained by chemical synthesis or PCR.

[0021] Preferably, the construction of a recombinant plasmid containing the nucleic acid molecule encoding human defensin described in the first aspect in step (1) comprises: synthesizing the nucleic acid molecule encoding human defensin, replacing the his tag and the thrombin cleavage site located after the lytic tag TrxA in the plasmid with a high-specificity tag and a cleavage site without amino acid residues, removing the tag used for high protein purification and the tag without amino acid residues, adding 5' (MscI) and 3' (XhoI), and cloning the nucleic acid molecule encoding human defensin into a vector to obtain a recombinant plasmid.

[0022] Preferably, the highly specific tag comprises twins-strep.

[0023] Preferably, the cleavage site without amino acid residues includes an enterokinase cleavage site.

[0024] Preferably, the purification in step (3) includes ultrafiltration and enzymatic digestion.

[0025] Preferably, the enzymatic cleavage includes: washing the human defensin fusion protein bound to the purification column with an enzymatic cleavage buffer, resuspending the human defensin fusion protein purification column with an enzymatic cleavage buffer, adding enterokinase containing a his tag, enzymatic cleavage overnight, collecting the flow-through, removing enterokinase, and centrifuging the flow-through to obtain human defensin.

[0026] It is understood that the expression system in the present invention also includes expression systems well known to those skilled in the art, such as plant expression systems, insect cell expression systems (Baculovirus expression), mammalian cell expression systems, cell-free protein expression systems or yeast expression systems.

[0027] In a fifth aspect, the present invention provides a human defensin, which is prepared by the recombinant preparation method of human defensin described in the fourth aspect.

[0028] It is understood that the human defensins and their functionally equivalent mutants and derivatives (such as modified versions and pharmaceutically acceptable salts) in the present invention are all within the scope of protection of the present invention.

[0029] In the sixth aspect, the present invention provides a fusion protein, which contains the human defensin described in the fifth aspect, and the fusion protein contains the human defensin and collagen binding domain described in claim 7, the human defensin and collagen binding domain in the fusion protein are connected by a flexible connecting peptide, and the collagen binding domain is located at the N-terminus or C-terminus of the fusion protein.

[0030] Preferably, the amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.3.

[0031] In the present invention, the fusion protein can specifically bind to the collagen membrane scaffold and achieve sustained release. The collagen membrane scaffold includes a scaffold material with type 1 collagen, such as an acellular matrix collagen scaffold, a collagen / gelatin electrospinning membrane scaffold or a gelatin methacrylated hydrogel scaffold.

[0032] In the present invention, the fusion protein can be used for the preparation of biomedical materials, and the biomedical materials contain the fusion protein of the present invention and acellular collagen membrane.

[0033] In the present invention, the method for preparing the biomedical material comprises the following steps:

[0034] (1) constructing an expression vector encoding the fusion protein and expressing it in a host cell;

[0035] (2) Purification to obtain the fusion protein;

[0036] (3) The fusion protein solution is applied to the acellular collagen membrane to form a sustained-release system.

[0037] The acellular collagen membrane of the present invention can sustainably release the fusion protein in PBS for more than 7 days.

[0038] SEQ ID NO.3:

[0039] TKKTLRTGGGGGSGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0040] Preferably, the nucleic acid sequence encoding the fusion protein includes any one of the following:

[0041] (1) the sequence shown in SEQ ID NO. 4;

[0042] (2) A nucleic acid sequence encoding a human defensin obtained by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO. 4;

[0043] (3) A nucleic acid sequence that has at least 80% sequence homology with the nucleic acid sequence described in (1) or (2) and has the same or similar functions.

[0044] SEQ ID NO.4:

[0045] ACAAAGAAAACATTACGGACCGGTGGAGGCGGTTCAGGCATTATCAACACCCTGCAGAAATATTACTGCCGCGTGAGGGGAGGGAGATGTGCAGTGTTGAGTTGCTTACCAAAAGAAGAACAGATAGGGAAATGTAGTACCAGAGGAAGGAAATGTTGTAGAAGGAAGAAA.

[0046] In a seventh aspect, the present invention provides the use of the nucleic acid molecule encoding human defensin described in the first aspect, the vector described in the second aspect, the recombinant cell described in the third aspect, or the human defensin described in the fifth aspect in the preparation of antibacterial, anti-inflammatory or tissue regeneration products.

[0047] Preferably, the product comprises any one of a drug, a healing material or a medical device coating.

[0048] In this study, molecular biological experiments have clarified the mechanism by which hBD-3 acts on fibroblasts. It primarily inhibits the binding of TSP-1 to CD36, reducing TGF-β1 production. It selectively acts on both normal and activated fibroblasts, thereby reducing scar formation, providing a novel mechanistic basis for tissue repair. This novel function has broad applicability across various models (e.g., wound healing models and mucosal injury models). hBD-3 has demonstrated significant efficacy in wound healing (skin trauma, burns) and tissue repair (mucosal injury, corneal repair). It has potential applications in regenerative medicine (e.g., postoperative tissue repair and graft healing) and aesthetic medicine (e.g., skin regeneration).

[0049] The hBD-3 recombinant protein prepared by the present invention can be applied not only to animal models, but also to ultraviolet-treated fibroblast models, providing new insights into the molecular basis of ultraviolet-induced skin damage and potential strategies for skin repair and photoprotection.

[0050] Chronic or excessive UV exposure can lead to a series of skin changes, including inflammation, DNA damage, oxidative stress, and extracellular matrix (ECM) remodeling, ultimately leading to photoaging, pigmentation disorders, and even tumorigenesis. Dermal fibroblasts are the main ECM-producing cells in the dermis and play a key role in maintaining skin structure and function. Fibroblasts are highly sensitive to UV-induced stress responses and undergo significant phenotypic and functional changes after irradiation. Low-dose UV can induce stress activation, simulating the damage caused by UV rays in sunlight to the skin dermis. Therefore, the UV-treated fibroblast model provides a valuable platform for exploring the cellular and molecular mechanisms of UV-induced skin damage and evaluating the efficacy of photoprotective agents.

[0051] The hBD-3 recombinant protein obtained in the present invention can achieve functions such as extracellular matrix remodeling, angiogenesis, scar inhibition and cell proliferation. In addition, it has other potential applications such as promoting skin repair, enhancing skin barrier function and improving skin appearance.

[0052] hBD-3 can be prepared into a variety of pharmaceutical forms, including topical ointments, sprays, injections, etc.

[0053] Both genetically engineered expression systems and modified versions (such as functional mutants) of hBD-3 can be applied to therapeutic product development.

[0054] In the present invention, hBD-3 exhibits strong thermal stability and biological activity, maintaining its function in a variety of environments. As an endogenous protein, hBD-3 is highly safe for use and has a low risk of toxic side effects.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The present invention significantly improves the stability of hBD-3 by fusing it with Trx A, reducing the loss caused by degradation during the expression process;

[0057] (2) The method of the present invention constructs a recombinant expression vector capable of recombinantly expressing human defensin hBD-3, releasing the recombinant fusion protein into the culture supernatant, facilitating protein purification. By fusing the Strep tag, affinity purification technology can be used to significantly simplify the protein purification process, and the number of purification times can be reduced by on-column enzyme cleavage, thereby improving the yield and purity of the target protein. The presence of the fusion tag enables the effective removal of non-specific proteins during the purification process, thereby obtaining high-purity hBD-3.

[0058] (3) The method of the present invention improves the solubility of the protein and reduces the formation of inclusion bodies. Since hBD-3 contains three pairs of disulfide bonds, inclusion bodies are easily formed during the expression process. By fusing it with certain highly soluble proteins (Trx A), the soluble expression of hBD-3 can be significantly improved, the formation of insoluble inclusion bodies can be reduced, and the active yield of the protein can be increased. In addition, by double mutation of thioredoxin reductase (trxB) and glutathione reductase (gor), the intracellular reduction pathway is destroyed, the oxidative environment is enhanced, and thus the formation of disulfide bonds is promoted. This mutation makes the cytoplasm closer to the oxidative state of eukaryotic cells, which is suitable for expressing proteins that rely on correct disulfide bonds. Origami strains are often used in combination with pET32a(+) vectors carrying thioredoxin (Trx) tags. The Trx tag acts as a molecular chaperone to further assist protein folding and inhibit aggregation, significantly improving the active expression efficiency of disulfide bond proteins.

[0059] (4) The present invention discovered for the first time that hBD-3 reduces the production of TGF-β1 by inhibiting the binding of TSP-1 to CD36, thereby reducing the formation of scars, and can selectively act on normal and activated fibroblasts, thus having broad application prospects in tissue repair and regenerative medicine. The recombinant human defensin obtained by the method of the present invention is beneficial to the development of multifunctional fusion proteins. By designing and fusing with other biologically active proteins, the multifunctional application of hBD-3 can be realized, such as in the fields of anti-infection coatings, wound dressings and tissue engineering scaffolds;

[0060] (5) The method of the present invention is suitable for large-scale production, is economical and efficient, and can be used to produce hBD-3 protein on a large scale in bacteria or other microorganisms through genetic recombination technology at low cost, making it suitable for industrial application;

[0061] (6) The method of the present invention is easy to operate, and the optimization of the fusion tag and expression system makes the method highly operable in both laboratory and production scales. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the plasmid constructed for the present invention;

[0063] Figure 2 This is the whole plasmid identification diagram;

[0064] Figure 3 The figure is the SDS-PAGE gel electrophoresis result in Example 2;

[0065] Figure 4 This is a diagram showing the results of the protein immunoblotting experiment in Example 2;

[0066] Figure 5 This is a diagram showing the results of the protein immunoblotting experiment in Example 3;

[0067] Figure 6 This is a graph showing the antibacterial test results in Example 4;

[0068] Figure 7 This is the qPCR result diagram in Example 5;

[0069] Figure 8 The agarose gel electrophoresis result in Example 5 is shown;

[0070] Figure 9A This is a heat map of fibroblast phenotypic genes after UV irradiation;

[0071] Figure 9B Heat map of regeneration and scar gene expression after hBD-3 treatment;

[0072] Figure 9C Heat map of angiogenic gene expression after hBD-3 treatment;

[0073] Figure 10 This is the result diagram of TGF-β1 expression;

[0074] Figure 11 This is a diagram of wound healing results;

[0075] Figure 12 This is the statistical result diagram of wound healing area;

[0076] Figure 13 This is the result of H&E staining;

[0077] Figure 14 This is the result diagram of protein release in Example 8. DETAILED DESCRIPTION

[0078] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0079] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0080] Example 1

[0081] Construction of expression engineered bacteria Origami-pET32a(+) / hBD-3.

[0082] 1. Codon Optimization

[0083] The full sequence of human β-defensin 3 (hBD-3) was predicted in the NCBI gene database and found to have three disulfide bonds, linked and fixed by Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. The amino acid sequence of the mature polypeptide is as follows:

[0084] hBD-3 (SEQ ID NO. 1):

[0085] GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0086] After codon optimization in E. coli, the optimized coding nucleic acid sequence is as follows:

[0087] hBD-3 (SEQ ID NO. 2):

[0088] GGAATTATCAACACCCTGCAGAAATATTACTGCCGCGTGAGGGGAGGGAGATGTGCAGTGTTGAGTTGCTTACCAAAAGAAGAACAGATAGGGAAATGTAGTACCAGAGGAAGGAAATGTTGTAGAAGGAAGAAA.

[0089] 2. Construction of pET32a(+) / hBD-3 recombinant plasmid

[0090] The gene hBD-3 was synthesized, and the his tag and thrombin cleavage site located after the lytic tag TrxA in the plasmid were replaced by twins-strep and enterokinase cleavage sites through genetic engineering methods for high-level protein purification and removal of tags without amino acid residues. 5' (MscI) and 3' (XhoI) were added, and the gene was cloned into the vector pET32a (+) (Ampicillin) through 5'MscI and 3'XhoI (using a recombination method) to construct a plasmid, such as Figure 1 shown.

[0091] 3. Construction of expression engineered bacteria Origami-pET32a(+) / hBD-3

[0092] (1) Preparation of competent cells: Escherichia coli Origami was prepared into competent cells using a one-step competent bacteria preparation kit (Biyuntian).

[0093] (2) Transformation experiment: The pET32a(+) / hBD-3 plasmid was transformed into E. coli Origami competent cells using conventional methods. The transformation product was inoculated on LB plates containing ampicillin and cultured overnight. The next day, positive colonies were picked and cultured for full plasmid detection and identification.

[0094] 4. Experimental results

[0095] The whole plasmid was tested and identified to have the correct sequence of hBD-3 mature peptide inserted into the plasmid without mutation ( Figure 2 ).

[0096] Example 2

[0097] Inducible expression and identification of hBD-3 recombinant protein.

[0098] 1. Inducible expression of hBD-3 recombinant protein

[0099] The expression strain Origami-pET32a(+) / hBD-3 stored in glycerol was inoculated into 5 mL LB / AMP liquid medium and cultured with shaking at 37°C overnight. 5 mL of the bacterial solution was re-inoculated into 1 L LB / AMP liquid medium at a 1:200 inoculum and cultured with shaking at 37°C and 200 rpm for 2 h until the bacteria reached the logarithmic growth phase (OD 600 =0.4-0.6). A positive control was made without IPTG, while the remaining cells were supplemented with IPTG to a final concentration of 0.2 mM. Culture was continued overnight at 18°C, and the supernatant was removed by centrifugation. The cells were resuspended in loading buffer (20 mM Tris, 150 mM NaCl, pH 7.6) for 1 g of bacteria (9 mL of loading buffer). Iron Hammer Ultra Bacteria Lysis Solution (ACE) was added to 1 / 9 the volume of the resuspension, and the suspension was mixed by stirring or vortexing. The sample was lysed at 25°C for 10 min, and the whole suspension was retained. The supernatant was then removed and the pellet was resuspended in an equal volume of loading buffer.

[0100] 2. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

[0101] The above samples (whole solution, precipitate, and supernatant) were added to 4× LDS protein loading buffer, boiled at 100°C for 10 minutes, and cooled for later use. Using an ACE gradient gel (4-20%), 30 μL of the prepared protein sample and 10 μL of protein marker were loaded into the sample wells. Electrophoresis conditions were: a voltage of 160 V for approximately 40 minutes. After electrophoresis, the gel was stripped and stained with Coomassie Brilliant Blue for 1 hour. After several rinses with Milli-Q water, the gel was destained three times with elution buffer (50 mL of methanol, 100 mL of glacial acetic acid, and 850 mL of Milli-Q water) on a shaker until the protein bands were clear. Electrophoresis results were recorded using a gel imager.

[0102] 3. Western blot identification

[0103] 30 μL of the above samples (whole solution, precipitate, supernatant) were added to 10 μL of 4×LDS protein electrophoresis loading buffer and boiled for 10 min. SDS-PAGE electrophoresis was performed. After the electrophoresis, the protein bands on the gel were transferred to a PVDF membrane and incubated with rabbit polyclonal antibody hBD-3 (1:1000) and goat anti-rabbit IgG antibody (1:4000) respectively. The membrane was developed with ECL luminescent solution and the image was retained.

[0104] 4. Experimental Results

[0105] Results of hBD-3 recombinant protein induction expression: SDS-PAGE gel electrophoresis results showed that after IPTG induction, an hBD-3 recombinant protein expression band appeared at about 25 KDa in the Origami-pET32a(+) / hBD-3 prokaryotic expression engineering bacteria, and no obvious expression of the target protein was observed after expression without IPTG induction. The results of SDS-PAGE gel electrophoresis after cleavage of the hBD-3 recombinant protein showed that ( Figure 3 ), most of the proteins obtained by IPTG-induced expression are soluble proteins and do not require denaturation and renaturation treatment.

[0106] Western blotting results of hBD-3 recombinant protein: Western blotting results showed that when rabbit polyclonal antibody hBD-3 was used as primary antibody, Origami-pET32a(+) / hBD-3 had a protein blotting band of about 25 KDa ( Figure 4 ).

[0107] Example 3

[0108] Purification and enzyme digestion of hBD-3 recombinant protein.

[0109] 1. Protein Purification

[0110] The supernatant of the bacterial solution obtained in Example 2 was used to purify the expressed hBD-3 recombinant protein according to the instructions of the Strep purification column of Kangti Life Science Company without elution.

[0111] 2. Enzymatic cleavage of recombinant proteins

[0112] On-column enzymatic digestion: After repeatedly washing the hBD-3 recombinant protein bound to the purification column with enzymatic digestion buffer, resuspend the column in enzymatic digestion buffer and add enterokinase (containing a His tag, final concentration 0.2 U / mL) to mix thoroughly. Digestion should proceed overnight at 25°C. The flow-through should be collected and enterokinase should be removed according to the instructions for the His purification magnetic beads from Yisheng Company. The flow-through should then be centrifuged at 12,000 × g for 20–40 min in an ultrafiltration centrifuge tube (3 kDa). The contents of the tube should be discarded and topped up with sterile Milli-Q water. Repeat the centrifugation two to three times to remove salts. Finally, the contents of the tube should be collected and quantified using a BCA protein quantification kit. The quantified recombinant hBD-3 polypeptide should be analyzed by SDS-PAGE electrophoresis and western blotting.

[0113] 3. Experimental Results

[0114] SDS-PAGE electrophoresis of the recombinant hBD-3 polypeptide showed that the tag protein was completely removed after digestion with enterokinase. The digestion product was purified, desalted, and concentrated to obtain hBD-3 (approximately 8 KDa). Western blotting of the recombinant hBD-3 protein revealed a protein band of approximately 8 KDa, which was consistent with the theoretical molecular weight ( Figure 5 ).

[0115] Example 4

[0116] Anti-E. coli activity of recombinant hBD-3.

[0117] 1. Preparation of Bacteria

[0118] Resuscitated E. coli strains were spread on agar plates, and after overnight culture, single clones were picked and inoculated into LB medium for expansion culture. The concentration of E. coli solution was adjusted to 10 7 CFU / mL, and blow evenly to prepare a bacterial suspension.

[0119] 2. Antibacterial detection

[0120] The antibacterial activity of hBD-3 was detected by agar plate diffusion method. 7CFU / mL, 0.1 mL) was inoculated onto solid agar medium. Sterile filter paper (6 mm diameter) saturated with hBD-3 (20 μg / mL) and blank LB were used as controls. The filter paper was placed on solid agar medium and incubated at 37°C for 24 h. The diameter of the growth inhibition zone ( Figure 6 ), indicating that the recombinant hBD-3 of the present invention can effectively inhibit bacteria.

[0121] Example 5

[0122] Application of hBD-3 in activating fibroblasts.

[0123] 1. Construction of activated fibroblasts

[0124] Fibroblasts were activated by ultraviolet irradiation. Cells were irradiated with c-wave (shortwave) ultraviolet light for 2 minutes and then irradiated for another 2 minutes after passage. After two irradiations, normal fibroblasts in the control group and activated fibroblasts in the irradiation group were obtained. qPCR and Western blotting were performed to characterize the fibroblast phenotype.

[0125] 2. hBD-3 intervention of different fibroblasts

[0126] NIH3T3 cells were evenly seeded in 96-well plates at 5000 cells / well. After 12 h of culture, 1 μg / mL hBD-3 was added to each experimental group. Cells were collected 24 h later and transcriptomic sequencing was performed. N represents normal fibroblasts, S represents activated fibroblasts, NH and SH represent normal fibroblasts + hBD-3 and activated fibroblasts + hBD-3.

[0127] 3. Experimental Results

[0128] qPCR ( Figure 7 ) and Western blotting ( Figure 8 ) The experiment verified the high expression of α-SMA and CD36, proving the successful construction of activated fibroblasts.

[0129] Transcriptomics was used to identify UV-irradiated fibroblasts, and the results showed that genes related to the activated fibroblast phenotype were significantly increased, indicating that the activated fibroblasts were successfully constructed ( Figure 9A Further analysis revealed that hBD-3 intervention caused normal fibroblasts to produce more proteins related to cell migration, adhesion, and actin polymerization, while activated fibroblasts produced fewer related proteins ( Figure 9B This suggests that hBD-3 can selectively intervene in fibroblasts to promote tissue regeneration. In addition, transcriptomic analysis found that genes related to angiogenesis factors in activated fibroblasts were upregulated, and the same trend was observed after hBD-3 intervention ( Figure 9C).

[0130] Example 6

[0131] Application of hBD-3 in scar inhibition.

[0132] 1. Mechanism Exploration

[0133] Since TSP-1 significantly increases after injury, it can bind to CD36 and produce TGF-β1. Therefore, in this experiment, TSP-1 (20 μg / mL) and hBD-3 (10 μg / mL) were introduced into normal fibroblasts and activated fibroblasts, and the production of TGF-β1 was observed.

[0134] 2. Experimental Results

[0135] The results showed that TGF-β1 was highly expressed in activated fibroblasts, and TSP-1 induced high expression of TGF-β1 in activated fibroblasts. hBD-3 reduced the expression of TGF-β1 in TSP-1 induced activated fibroblasts, and hBD-3 treated TSP-1 induced normal fibroblasts with no significant difference in TGF-β1 expression ( Figure 10 ).

[0136] Example 7

[0137] Application of hBD-3 in wound healing of rat skin.

[0138] A thermosensitive hydrogel was prepared using poloxamer 188, poloxamer 407, and hydroxypropyl methylcellulose. hBD-3 was added at a concentration of 1 μg / mL and administered once daily to a rat skin wound model with a wound diameter of 5 mm. In the rat skin wound model, hBD-3 (1 μg / mL) significantly accelerated wound healing after 7 days of application ( Figure 11 and Figure 12 ), and H&E staining showed that the epithelial tissue of rats in the hBD-3 group was able to proliferate rapidly and was no different from normal tissue, with reduced granulation tissue. Masson staining showed that the production of collagen in the regenerated epithelium of the hBD-3 group was similar to that of normal tissue ( Figure 13 ).

[0139] Example 8

[0140] Binding and release of CBD-hBD-3 fusion protein to collagen membrane (acellular matrix collagen).

[0141] 1. Preparation of CBD-hBD-3 Fusion Protein

[0142] The preparation method is the same as that of hBD-3 recombinant protein. Referring to Example 1, the amino acid sequence of the fusion protein CBD-hBD-3 is shown in SEQ ID NO.3. After codon optimization in E. coli, the optimized encoding nucleic acid sequence is shown in SEQ ID NO.4.

[0143] SEQ ID NO.3:

[0144] TKKTLRTGGGGGSGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0145] SEQ ID NO.4:

[0146] ACAAAGAAAACATTACGGACCGGTGGAGGCGGTTCAGGCATTATCAACACCCTGCAGAAATATTACTGCCGCGTGAGGGGAGGGAGATGTGCAGTGTTGAGTTGCTTACCAAAAGAAGAACAGATAGGGAAATGTAGTACCAGAGGAAGGAAATGTTGTAGAAGGAAGAAA.

[0147] 2. Binding of CBD-hBD-3 to Acellular Collagen Matrix

[0148] Different concentrations of CBD-hBD-3 (2.5, 5, 10, 20, 40 μg / cm 2 The solution ((CBD-hBD-3) is incubated with the decellularized matrix collagen at 4°C overnight. The remaining CBD-hBD-3 content in the supernatant is detected to calculate the binding amount of the decellularized matrix collagen and CBD-hBD-3.

[0149] 3. Release of CBD-hBD-3 on Acellular Collagen Matrix

[0150] The maximum concentration of CBD-hBD-3 (26.69 ± 1.19 μg / cm 2 The solution ((CBD-hBD-3) solution, which refers to the amount of CBD-hBD-3 co-incubated per square centimeter of decellularized matrix collagen) was incubated with the collagen membrane. After four days of overnight, the membrane was washed three times with PBS and fresh PBS was added. The protein content in the supernatant was detected on the 1st, 4th, and 7th days, and the protein release amount after 7 days was calculated.

[0151] 4. Experimental Results

[0152] The results are as follows Figure 14As shown in Figure 2, the maximum binding capacity of acellular matrix collagen was 26.69 ± 1.19 μg / cm 2 , and the cumulative release amount over seven days was 6.49 ± 0.63 μg, indicating that the fusion protein CBD-hBD-3 of the present invention can effectively bind to the collagen membrane (acellular matrix collagen) and achieve the purpose of sustained release.

[0153] In summary, the present invention constructs a recombinant expression vector capable of recombinantly expressing human defensin hBD-3, releasing the recombinant fusion protein into the culture supernatant, facilitating protein purification. The recombinant human defensin obtained by the method of the present invention has tissue regeneration function, providing a new active molecule for the development of new drugs.

[0154] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein contains human β-defensin 3. The human β-defensin 3 and the collagen binding domain in the fusion protein are connected by a flexible connecting peptide. The collagen binding domain is located at the N-terminus of the fusion protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO.

3.

2. The fusion protein according to claim 1, wherein The coding sequence of the fusion protein is shown in SEQ ID NO.

4.

3. The fusion protein according to claim 1, wherein The fusion protein can specifically bind to the collagen membrane scaffold and achieve sustained release.

4. The fusion protein according to claim 3, wherein The collagen membrane scaffold includes any one of a decellularized matrix collagen scaffold, a collagen / gelatin electrospinning membrane scaffold or a gelatin methacrylated hydrogel scaffold.

5. Use of the fusion protein according to any one of claims 1 to 4 in the preparation of antibacterial, anti-inflammatory or tissue regeneration products.

6. The use according to claim 5, characterized in that The product includes any one of a drug, a healing material or a medical device coating.

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