Nucleic acid molecule for coding human defensin as well as method for preparing human defensin and application of nucleic acid molecule

Abstract

The invention belongs to the fields of gene engineering, bioengineering and biological medicine, and relates to a nucleic acid molecule for coding human defensin, a method for preparing the human defensin and application. The nucleic acid sequence of the nucleic acid molecule comprises any one of the following sequences: (1) a sequence as shown in SEQ ID NO.2; (2) a nucleic acid sequence for coding human defensin, which is obtained by substituting, deleting or adding one or more than two nucleotides to the sequence as shown in SEQ ID NO.2; and (3) a nucleotide sequence which has at least 80% sequence homology with the nucleotide sequence in (1) or (2) and has the same or similar functions with the nucleotide sequence in (1) or (2). According to the method provided by the invention, the recombinant expression vector is constructed by using nucleic acid molecules for coding the human defensin, the human defensin hBD-3 can be recombined and expressed, the recombinant fusion protein is released to the supernatant of a culture solution, the protein purification is convenient, the obtained recombinant human defensin has a tissue regeneration function, and a new active molecule is provided for the development of a novel drug.

Description

Technical Field

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

[0002] Human beta-defensin 3 (hBD-3) is an antibacterial peptide naturally present in the human body and belongs to the beta-defensin family. This class of peptides mainly participates in the innate immunity of the body and resists infections by directly killing microorganisms and regulating the immune system. hBD-3 has broad-spectrum antibacterial activity and can not only effectively resist bacteria (including Gram-positive and Gram-negative bacteria) and fungi, but also inhibit certain viruses (such as human immunodeficiency virus, HIV). hBD-3 achieves its antibacterial effect by disrupting the cell membrane of pathogens. It can interact with the anionic membrane of pathogens, resulting in the rupture of the cell membrane and the death of pathogens.

[0003] Due to its strong antibacterial, immunomodulatory and tissue regeneration functions, hBD-3 has been widely studied for use in anti-infection therapy, wound healing promotion, and oral mucosa and vaginal regeneration. In the context of the increasingly serious problem of antibacterial drug resistance, hBD-3 represents a research direction with great potential as an alternative to antibiotics or adjuvant therapy. In short, due to its strong antibacterial and immunomodulatory effects, as well as its unique advantages in promoting tissue regeneration and low induction of drug resistance, hBD-3 has become an important focus for future biomedical research applications. However, the content of human defensin in organisms is low and extraction is difficult, while chemical synthesis methods are costly. Therefore, using genetic engineering means is an economical and effective way to mass-produce defensin. However, when expressing the complex eukaryotic protein hBD-3, it is prone to misfolding and formation of inclusion bodies in Escherichia coli, and the refolding efficiency is low, resulting in low expression yield and activity of functional proteins. Therefore, it is difficult to recombinantly prepare human defensin using prokaryotes, and it is also difficult to guarantee the antibacterial activity of recombinant polypeptides. This is the key factor restricting the recombinant preparation of defensin at present.

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

[0005] In view of the deficiencies of the prior art and the 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 the recombinant expression vector of human defensin based on the pET-32a(+) vector framework, transforms it into Escherichia coli competent Origami, and explores the expression conditions through small-scale protein experiments and SDS-PAGE gel electrophoresis. It is found that human defensin and the pET-32a(+) recombinant expression vector are in the supernatant, and the fusion protein Trx-hBD-3 is successfully expressed. The fusion protein is cleaved by enterokinase, and then the protein is purified according to the expressed protein tag, and human defensin is successfully obtained. The recombinant human defensin of the present invention has various functions such as antibacterial and promoting collagen expression, and has potential application value.

[0006] To achieve the object of the present invention, the following technical solutions are adopted:

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

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

[0009] (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;

[0010] (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.

[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 the function of tissue regeneration and provides a new active molecule for the development of new drugs.

[0012] SEQ ID NO.2:

[0013] GGAATTATCAACACCCTGCAGAAATATTACTGCCGCGTGAGGGGAGGGAGATGTGCAGTGTTGAGTTGCTTACCAAAAGAAGAACAGATAGGGAAATGTAGTACCAGAGGAAGGAAATGTTGTAGAAGGAAGAAA.

[0014] In the second aspect, the present invention provides a vector, and the vector contains the nucleic acid molecule encoding human defensin described in the first aspect.

[0015] In a third aspect, the present invention provides a recombinant cell, which contains the nucleic acid molecule encoding human defensin described in the first aspect or the vector described in the second aspect.

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

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

[0018] (2) Use an inducer to induce the human defensin expression strain to express and obtain a 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 the recombinant plasmid containing the nucleic acid molecule encoding human defensin described in the first aspect in step (1) includes: synthesizing the nucleic acid molecule encoding human defensin, modifying and replacing the his tag and thrombin cleavage site after the solubility-enhancing tag TrxA in the plasmid with a highly specific tag and a tag-free amino acid residue cleavage site, removing the tag for highly purifying the protein and the tag-free amino acid residue, adding 5'(MscI) and 3'(XhoI), and cloning the nucleic acid molecule encoding human defensin into the vector to obtain a recombinant plasmid.

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

[0023] Preferably, the tag-free amino acid residue cleavage site includes enterokinase cleavage site.

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

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

[0026] It can be 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] Fifth aspect, the present invention provides a human defensin, which is prepared by the recombinant preparation method of the human defensin described in the fourth aspect.

[0028] It can be understood that the human defensin, mutants and derivatives with equivalent functions (such as modified versions, pharmaceutically acceptable salts) in the present invention are all within the protection scope of the present invention.

[0029] Sixth aspect, the present invention provides a fusion protein, which contains the human defensin described in the fifth aspect, the fusion protein contains the human defensin described in claim 7 and a collagen-binding domain, and the human defensin and the collagen-binding domain in the fusion protein are connected by a flexible linker 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 acellular matrix collagen scaffolds, collagen / gelatin electrospun membrane scaffolds, or gelatin methacryloylated hydrogel scaffolds and other scaffold materials with type I collagen.

[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 described in the present invention and an acellular collagen membrane.

[0033] In the present invention, the preparation method of the biomedical material includes the following steps:

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

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

[0036] (3) Apply the fusion protein solution to the acellular collagen membrane to make it bind to form a sustained release system.

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

[0038] SEQ ID NO.3:

[0039] TKKTLRTGGGGSGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0040] Preferably, the coding nucleic acid sequence of 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 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 having at least 80% sequence homology with the nucleic acid sequence described in (1) or (2) and having the same or similar function.

[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 products for antibacterial, anti-inflammatory or tissue regeneration.

[0047] Preferably, the product includes any one of drugs, healing materials or medical device coatings.

[0048] In the present invention, through molecular biology experiments, the mechanism of hBD-3 acting on fibroblasts was clarified. It mainly reduces the generation of TGF-β1 by inhibiting the binding of TSP-1 and CD36, and can selectively act on normal and activated fibroblasts, thereby reducing the generation of scars, providing a new mechanism basis for tissue repair. This new function has broad applicability in different types of models (such as wound healing models, mucosal damage, etc.). hBD-3 has significant effects in the fields of wound healing (skin trauma, burns), tissue repair (mucosal damage, corneal repair), etc. It has potential applications in regenerative medicine (such as postoperative tissue repair, graft healing) and the aesthetic medicine field (such as skin regeneration).

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

[0050] Long-term or excessive ultraviolet (UV) irradiation can lead to a series of skin changes, including inflammation, DNA damage, oxidative stress, and extracellular matrix (ECM) remodeling, ultimately resulting in photoaging, pigment disorders, and even tumorigenesis. Dermal fibroblasts are the main cells in the dermis that produce ECM and play a key role in maintaining skin structure and function. Fibroblasts are highly sensitive to UV-induced stress and undergo significant phenotypic and functional changes after irradiation. Low-dose UV can induce stress activation and mimic the damage to the skin dermis layer by UV in sunlight. Therefore, the UV-treated fibroblast model is 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 ECM remodeling, angiogenesis, scar inhibition, and cell proliferation. In addition, there are other potential applications such as promoting skin repair, enhancing skin barrier function, and improving skin appearance.

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

[0053] The genetic engineering expression system and modified versions (such as functional mutants) of hBD-3 can both be applied to the development of therapeutic products.

[0054] In the present invention, hBD-3 exhibits strong thermal stability and biological activity and can maintain its function in various environments. As an endogenous protein, hBD-3 has high application safety and a low risk of toxic and side effects.

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

[0056] (1) By fusing with Trx A in the method of the present invention, the stability of hBD-3 is significantly improved, reducing the loss caused by degradation during the expression process;

[0057] (2) The recombinant expression vector constructed by the method of the present invention can recombinantly express human defensin hBD-3, release the recombinant fusion protein into the culture supernatant, facilitate protein purification. By fusing the Strep tag, the protein purification process can be significantly simplified using affinity purification technology, and the number of purification steps can be reduced by on-column digestion, improving the yield and purity of the target protein. The presence of the fusion tag enables effective removal of non-specific proteins during the purification process, 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, it is prone to form inclusion bodies during the expression process. By fusing with some highly soluble proteins (Trx A), the soluble expression of hBD-3 can be significantly improved, the generation 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 disrupted, 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 and is suitable for expressing proteins that rely on correct disulfide bonds. The Origami strain is often used in combination with the pET32a(+) vector carrying the thioredoxin (Trx) tag. The Trx tag acts as a molecular chaperone, further assisting protein folding and inhibiting aggregation, and significantly improving the active expression efficiency of disulfide bond proteins;

[0059] (4) The present invention discovers for the first time that hBD-3 reduces the generation 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 to fuse with other bioactive proteins, the multifunctional application of hBD-3 can be realized, such as applications 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, economical and efficient. Through genetic recombination technology, hBD-3 protein can be mass-produced in bacteria or other microorganisms, with low cost and suitable for industrial application;

[0061] (6) The method of the present invention is easy to operate. The optimization of the fusion tag and expression system makes this method highly operable both in the laboratory and on a production scale. Description of the Drawings

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

[0063] Figure 2 Full plasmid sequencing identification diagram;

[0064] Figure 3 SDS-PAGE gel electrophoresis result diagram in Example 2;

[0065] Figure 4 Protein immunoblotting experiment result diagram in Example 2;

[0066] Figure 5 Protein immunoblotting experiment result diagram in Example 3;

[0067] Figure 6 It is the bacteriostatic detection result graph in Example 4;

[0068] Figure 7 It is the qPCR result graph in Example 5;

[0069] Figure 8 It is the agarose gel electrophoresis result graph in Example 5;

[0070] Figure 9A It is the heat map of fibroblast phenotype genes after ultraviolet irradiation;

[0071] Figure 9B It is the heat map of gene expression of regeneration and scar after hBD-3 treatment;

[0072] Figure 9C It is the heat map of angiogenic gene expression after hBD-3 treatment;

[0073] Figure 10 It is the TGF-β1 expression result graph;

[0074] Figure 11 It is the wound healing result graph;

[0075] Figure 12 It is the statistical result graph of wound healing area;

[0076] Figure 13 It is the H&E staining result graph;

[0077] Figure 14 It is the protein release amount result graph in Example 8. Specific Embodiments

[0078] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention.

[0079] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0080] Example 1

[0081] Construction of the expression engineering bacterium Origami-pET32a(+) / hBD-3.

[0082] 1. Codon optimization

[0083] In the NCBI gene database, the full sequence information of human β-defensin 3 (hBD-3) was predicted. It has 3 pairs of disulfide bonds, which are fixed by the connection modes of Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. The amino acid sequence information of the mature polypeptide is as follows:

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

[0085] GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0086] Through codon optimization of Escherichia 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. The his tag and thrombin cleavage site after the solubilizing tag TrxA in the plasmid were modified and replaced with twins-strep and enterokinase cleavage site by genetic engineering methods for highly purifying the protein and removing the tag 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 the recombinant method) to construct the plasmid, as Figure 1 shown.

[0091] 3. Construction of the expression engineering bacterium Origami-pET32a(+) / hBD-3

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

[0093] (2)Transformation experiment: The pET32a(+) / hBD-3 plasmid was transformed into competent Escherichia coli Origami cells by conventional methods. The transformed products were inoculated on an LB plate containing ampicillin and cultured overnight. The next day, positive colonies were picked and cultured, and then the whole plasmid was sequenced for identification.

[0094] 4. Experimental results

[0095] After whole plasmid sequencing identification, the inserted hBD-3 mature peptide sequence in the plasmid was correct and there were no mutations ( Figure 2 ).

[0096] Example 2

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

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

[0099] The expression strain Origami-pET32a(+) / hBD-3 stored in glycerol was inoculated into 5 mL of LB / AMP liquid medium and cultured overnight at 37°C with shaking. Then, 5 mL of the bacterial solution was re-inoculated into 1 L of LB / AMP liquid medium at an inoculation ratio of 1:200 and cultured at 37°C with shaking at 200 rpm for 2 h until the bacteria reached the logarithmic growth phase (OD 600 = 0.4 - 0.6). As a positive control, IPTG was not added, and IPTG was added to the others to a final concentration of 0.2 mM, and then the culture was continued overnight at 18°C. After centrifugation to remove the supernatant, the bacterial cells were resuspended with loading buffer (20 mM Tris, 150 mM NaCl, pH = 7.6) (9 mL of loading buffer was added to 1 g of bacteria). Hammer Super Bacterial Lysis Solution (ACE) was added at 1 / 9 of the volume of the bacterial cell resuspension, and the mixture was stirred or shaken evenly. The sample was placed at 25°C for 10 min to lyse, and the whole solution was retained. Subsequently, it was centrifuged at 8000 rpm at 4°C for 20 min, and the supernatant was taken. The precipitate was resuspended with an equal volume of loading buffer.

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

[0101] Add the above samples (whole liquid, precipitate, supernatant) to 4×LDS protein loading buffer, boil at 100 °C for 10 min, and cool for later use. Load 30 μL of the prepared protein sample and 10 μL of protein molecular marker into the sample wells using a gradient gel (4-20%) from ACE Company. The electrophoresis conditions are as follows: the voltage of the sample in the gel is 160 V, and the electrophoresis time is about 40 min. After electrophoresis, strip the gel, add Coomassie Brilliant Blue staining solution, and stain for 1 h. After rinsing several times with Milli-Q water, perform three decolorizations on a shaker with the elution solution (50 mL of methanol + 100 mL of glacial acetic acid + 850 mL of Milli-Q water) until the protein bands are clear. Record the electrophoresis results using a gel imager.

[0102] 3. Identification by Western blot

[0103] Add 30 μL of the above samples (whole liquid, precipitate, supernatant) to 10 μL of 4×LDS protein electrophoresis loading buffer, boil for 10 min, perform SDS-PAGE electrophoresis. After electrophoresis, transfer the protein bands on the gel to a PVDF membrane, incubate successively with rabbit polyclonal antibody hBD-3 (1:1000) and goat anti-rabbit IgG antibody (1:4000), and develop with ECL luminescent solution to retain the image.

[0104] 4. Experimental results

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

[0106] Protein immunoblot identification results of hBD-3 recombinant protein: The protein immunoblot experiment results show that when using rabbit polyclonal antibody hBD-3 as the primary antibody, there is a protein blot band of about 25 KDa in Origami-pET32a(+) / hBD-3 Figure 4 ).

[0107] Example 3

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

[0109] 1. Protein purification

[0110] The supernatant of the bacterial liquid obtained in Example 2 was used to purify the expressed recombinant hBD-3 protein with reference to the instruction manual of the Strep purification column of Kangti Life Co., Ltd., without elution.

[0111] 2. Digestion of recombinant protein

[0112] On-column digestion: The recombinant hBD-3 protein bound to the purification column was repeatedly rinsed with digestion buffer, and then the hBD-3 recombinant protein purification column was resuspended with digestion buffer. Enterokinase (containing his tag, final concentration 0.2 U / mL) was added and mixed evenly, and digestion was carried out overnight at 25 °C. The flow-through was collected, and enterokinase was removed with reference to the instruction manual of the his purification magnetic beads of Yeasen Biotech Co., Ltd. Subsequently, the flow-through was centrifuged at 12,000×g for 20 - 40 min using an ultrafiltration centrifugal tube (3KDa). The liquid in the sleeve was discarded, and the liquid in the inner tube was supplemented with sterile Milli-Q water. The centrifugation was repeated 2 - 3 times to remove salts. Finally, the liquid in the inner tube was collected and quantified using a BCA protein quantification kit. The quantified recombinant hBD-3 polypeptide was identified by SDS-PAGE electrophoresis and Western blot.

[0113] 3. Experimental results

[0114] SDS-PAGE electrophoresis results of recombinant hBD-3 polypeptide: After digestion with enterokinase, the tag protein was completely removed. The digestion product was purified, desalted, and concentrated to obtain hBD-3 (about 8 KDa). Western blot identification results of recombinant hBD-3: The Western blot experiment results of the recombinant hBD-3 protein showed a protein blot band of about 8 KDa, and its molecular weight was consistent with the theoretical value ( Figure 5 )

[0115] Example 4

[0116] Antibacterial activity of recombinant hBD-3 against Escherichia coli

[0117] 1. Preparation of bacteria

[0118] The preserved Escherichia coli strain was revived, and the revived Escherichia coli was spread on an agar plate. After overnight culture, single colonies were picked and inoculated into LB medium for expanded culture. The concentration of the Escherichia coli bacterial liquid was adjusted to 10 7 CFU / mL, and it was blown evenly to prepare a bacterial suspension.

[0119] 2. Detection of antibacterial activity

[0120] The agar disc diffusion method was used to detect the antibacterial activity of hBD-3. The Escherichia coli bacterial suspension (10 7CFU / mL, 0.1 mL) were inoculated onto solid agar medium. Sterile filter paper discs (6 mm in diameter) saturated with hBD-3 (20 μg / mL) and blank LB were used as controls. The filter paper discs were placed on the solid agar medium and incubated at 37 °C for 24 h, and the diameter of the growth inhibition zone was measured ( 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 under ultraviolet irradiation. The cells were irradiated with c-wave (short-wave) ultraviolet light for 2 min, and then irradiated for another 2 min after subculture. After two irradiations, normal fibroblasts in the control group and activated fibroblasts in the irradiated group were obtained, and qPCR and Western blot experiments were performed to identify the phenotypes of the fibroblasts.

[0125] 2. Intervention of hBD-3 on different fibroblasts

[0126] 5000 NIH3T3 cells per well were evenly seeded in a 96-well plate. After culturing for 12 h, 1 μg / mL of hBD-3 was added to each group in the experimental group. After 24 h, the cells were collected for transcriptomic sequencing. 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 blot ( Figure 8 ) experiments verified the high expression of α-SMA and CD36, proving the successful construction of activated fibroblasts.

[0129] Identification of ultraviolet-irradiated fibroblasts by transcriptomics showed that genes related to the phenotype of activated fibroblasts were significantly upregulated, indicating the successful construction of activated fibroblasts ( Figure 9A ). Further analysis of the results found that the intervention of hBD-3 caused normal fibroblasts to produce more proteins related to cell migration, adhesion, and actin polymerization, while less related proteins were produced in activated fibroblasts ( Figure 9B ). This indicates that hBD-3 can selectively intervene in fibroblasts to promote tissue regeneration. In addition, transcriptomic detection found that genes related to pro-angiogenic factors in activated fibroblasts were upregulated, and hBD-3 intervention showed the same trend ( Figure 9C).

[0130] Example 6

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

[0132] 1. Mechanism exploration

[0133] Since TSP-1 is significantly increased after injury, which can bind to CD36 and produce TGF-β1, 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, 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 there was no significant difference in the expression of TGF-β1 in normal fibroblasts treated with hBD-3 induced by TSP-1 ( Figure 10 ).

[0136] Example 7

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

[0138] A thermosensitive hydrogel was prepared using poloxamer 188, poloxamer 407 and hydroxypropyl methylcellulose, and hBD-3 with a concentration of 1 μg / mL was added. Administration was carried out once a day in a rat skin model with a wound diameter of 5 mm. In the rat skin wound model, application of hBD-3 (1 µg / mL) significantly accelerated wound healing after 7 days ( Figure 11 and Figure 12 ), and it was observed by H&E staining that the epithelial tissue of rats in the hBD-3 group could proliferate rapidly and was indistinguishable from normal tissue, and the granulation tissue was reduced. 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 the 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. Through codon optimization of Escherichia coli, the optimized coding nucleic acid sequence is shown in SEQ ID NO.4.

[0143] SEQ ID NO.3:

[0144] TKKTLRTGGGGSGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0145] SEQ ID NO.4:

[0146] ACAAAGAAAACATTACGGACCGGTGGAGGCGGTTCAGGCATTATCAACACCCTGCAGAAATATTACTGCCGCGTGAGGGGAGGGAGATGTGCAGTGTTGAGTTGCTTACCAAAAGAAGAACAGATAGGGAAATGTAGTACCAGAGGAAGGAAATGTTGTAGAAGGAAGAAA.

[0147] 2. Binding of CBD-hBD-3 to decellularized matrix collagen

[0148] Solutions of different concentrations of CBD-hBD-3 (2.5, 5, 10, 20, 40 μg / cm 2 , referring to the amount of CBD-hBD-3 co-incubated with per square centimeter of decellularized matrix collagen) were co-incubated with decellularized matrix collagen overnight at 4°C. The content of the remaining CBD-hBD-3 in the supernatant was detected, and the binding amount of decellularized matrix collagen to CBD-hBD-3 was calculated.

[0149] 3. Release of CBD-hBD-3 from decellularized matrix collagen

[0150] The solution of the maximum concentration of CBD-hBD-3 that can be bound by decellularized matrix collagen (26.69 ± 1.19 μg / cm 2 , referring to the amount of CBD-hBD-3 co-incubated with per square centimeter of decellularized matrix collagen) was co-incubated with the collagen membrane overnight at 4°C. After that, it 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 in 7 days was calculated.

[0151] 4. Experimental results

[0152] The results are as Figure 14As shown, the maximum binding amount of acellular matrix collagen was 26.69 ± 1.19 μg / cm 2 , and the cumulative release amount in 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, which can recombinantly express human defensin hBD-3, 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 the function of tissue regeneration, providing a new active molecule for the development of new drugs.

[0154] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A nucleic acid molecule encoding a human defensin, characterized in that The nucleic acid sequence of the nucleic acid molecule includes any one of the following: (1) the sequence 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 that has at least 80% sequence homology with the nucleic acid sequence described in (1) or (2) and has the same or similar functions.

2. A carrier, characterized in that The vector contains the nucleic acid molecule encoding human defensin according to claim 1.

3. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule encoding human defensin according to claim 1 or the vector according to claim 2.

4. A method for recombinant preparation of human defensins, characterized in that: The recombinant preparation method comprises the following steps: (1) constructing a recombinant plasmid containing the nucleic acid molecule encoding human defensin according to claim 1, transforming it into competent cells, and culturing it to obtain a human defensin expression strain; (2) using an inducer to induce the human defensin expression strain to express a recombinant human defensin fusion protein; (3) Purify the recombinant human defensin fusion protein obtained in step (2).

5. The method for recombinant preparation of human defensins according to claim 4, characterized in that: The step (1) of constructing a recombinant plasmid containing the nucleic acid molecule encoding human defensin according to claim 1 comprises: synthesizing the nucleic acid molecule encoding human defensin, transforming and 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; The highly specific tags include twins-strep; The non-amino acid residue cleavage site includes an enterokinase cleavage site.

6. The method for recombinant preparation of human defensins according to claim 4, characterized in that: The purification in step (3) includes ultrafiltration and enzyme digestion; The enzyme digestion comprises: washing the human defensin fusion protein bound to the purification column with an enzyme digestion buffer, resuspending the human defensin fusion protein purification column with an enzyme digestion buffer, adding enterokinase containing a his tag, performing enzyme digestion overnight, collecting the flow-through, removing the enterokinase, and centrifuging the flow-through to obtain the human defensin.

7. A human defensin, characterized in that The human defensin is prepared by the recombinant preparation method of human defensin according to any one of claims 4-6.

8. A fusion protein, characterized in that The fusion protein contains the human defensin and collagen binding domain of claim 7, wherein 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; The fusion protein can specifically bind to the collagen membrane scaffold and achieve sustained release; 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; The amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.3; The nucleic acid sequence encoding the fusion protein includes any one of the following: (1) the sequence shown in SEQ ID NO.4; (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.4; (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.

9. Use of the nucleic acid molecule encoding human defensin according to claim 1, the vector according to claim 2, the recombinant cell according to claim 3, the human defensin according to claim 7 or the fusion protein according to claim 8 in the preparation of antibacterial, anti-inflammatory or tissue regeneration products.

10. The use according to claim 9, characterized in that: The product includes any one of a drug, a healing material or a medical device coating.

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