A full-groove hard tick-derived defensin polypeptide and uses thereof

Abstract

The application discloses a full-gorge hard tick-derived defensin polypeptide and application thereof. The defensin polypeptide is IpDf3, and the amino acid sequence is shown as SEQ ID NO:1. The defensin polypeptide has different degrees of antibacterial activity on four gram-positive bacteria and two gram-negative bacteria. Bactericidal kinetics and scanning electron microscope experiments show that 3*MIC IpDf3 can quickly down-regulate the bacterial number by 1-4 logarithmic units in 60 min, and can cause irregular bulges or depressions on the surface of gram-positive bacteria, and even rupture of the cell membrane, indicating that the antibacterial mechanism may be closely related to the direct damage of the cell membrane. The defensin polypeptide not only has the activity of traditional defensin against gram-positive bacteria, but also has certain antibacterial activity against gram-negative bacteria, has a wide antibacterial spectrum, strong activity, fast bactericidal speed, and is expected to provide a candidate for the research and development of new antibacterial drugs, and has important application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the gene discovery and research on the antibacterial activity and mechanism of the defensin polypeptide IpDf3 from *Ixodes persulcatus*. Background Technology

[0002] As an obligate hemophagocytic ectoparasite widely distributed globally, ticks are the second largest vector organism after mosquitoes. They can transmit various pathogens through bites or bloodsucking, seriously threatening human health and the development of animal husbandry. However, they themselves can remain uninfected during exposure to large numbers of pathogens, suggesting that they may be rich in special active substances that defend against pathogens. Defensins are a class of disulfide-rich cationic polypeptides widely distributed in fungi, plants, mammals, scorpions, mollusks, and ticks. They are important components of the innate immune system of organisms, exhibiting inhibitory activity against various microorganisms such as bacteria, viruses, and fungi, and possessing high research value and broad application prospects. Due to their unique antibacterial mechanism, defensins have become an important molecular framework for the development of novel antibacterial drugs. Meanwhile, bacterial resistance is a global public health problem, with most antibiotic-resistant infections caused by ESKAPE pathogens (staphylococci, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae). Insects are diverse and contain abundant defensin resources. Their unique polypeptide structure and natural broad-spectrum antibacterial activity are expected to provide new strategies for the development of novel antibacterial drugs.

[0003] Therefore, there is an urgent need to develop new antibacterial drugs that can replace antibiotics, especially natural active substances derived from insects. Summary of the Invention

[0004] The purpose of this invention is to discover a novel antimicrobial defensin IpDf3 derived from *Ixodes persulcatus* and its bactericidal mechanism. It not only possesses the activity of traditional defensins against Gram-positive bacteria but also exhibits certain antimicrobial activity against Gram-negative bacteria. It has a broad antimicrobial spectrum, strong activity, and rapid bactericidal speed, and is expected to provide a candidate for the development of novel antimicrobial drugs, showing significant application potential. The present invention adopts the following technical solution: In a first aspect of the invention, a defensin polypeptide derived from *Ixodes persulcatus* is provided, said defensin polypeptide being IpDf3, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0005] Furthermore, specific structural information such as Figure 2As shown. The reduced IpDf3 with the amino acid sequence shown in SEQ ID NO: 1 was obtained by chemical synthesis. The oxidized IpDf3 polypeptide (Ox-IpDf3) was prepared by in vitro expression, which contains 3 pairs of intramolecular disulfide bonds formed by 6 cysteine ​​residues, with pairing modes of Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6.

[0006] In a second aspect of the invention, a nucleic acid molecule encoding the said polypeptide is provided.

[0007] In a third aspect of the invention, a recombinant expression vector containing the aforementioned nucleic acid molecule is provided.

[0008] In a fourth aspect of the invention, a recombinant bacterium or engineered host cell line comprising the recombinant expression vector is provided.

[0009] In a fifth aspect of the invention, a method for preparing the defensin polypeptide is provided, the method comprising: The gene fragment encoding the defensin polypeptide IpDf3 was cloned into a vector to obtain a recombinant vector; The recombinant vector was induced to express the protein, and the defensin polypeptide was obtained after purification.

[0010] As another implementation method, it can also be synthesized directly based on the amino acid sequence.

[0011] In a sixth aspect of the invention, the use of the polypeptide in the preparation of medicaments against Gram-positive and / or Gram-negative bacteria is provided.

[0012] Furthermore, the whole-sulcus tick defensin IpDf3 exhibits varying degrees of antibacterial activity against four Gram-positive bacteria and two Gram-negative bacteria.

[0013] The Gram-positive bacteria include at least one of Staphylococcus aureus and Staphylococcus epidermidis.

[0014] The Gram-negative bacteria include at least one of Escherichia coli ATCC8739 and Escherichia coli ATCC25922. Furthermore, the whole-sulcus tick defensin IpDf3 exhibits the lowest MIC (minimum inhibitory concentration) against Gram-positive bacteria S. aureus AB94004 and S. epidermidis AB208188, approximately 0.0625 μM; it also shows some antibacterial activity against E. coli ATCC25922 and E. coli ATCC8739, with MIC values ​​around 8 μM.

[0015] Furthermore, the 3×MIC IpDf3 rapidly downregulated the colony count by 1-4 log units within 60 min.

[0016] Furthermore, the defensin IpDf3 can cause irregular protrusions or depressions on the surface of Gram-positive and Gram-negative bacteria after 30 min. After 2 h of treatment, the morphological changes are severe, with irregular protrusions and depressions appearing on the surface of the bacteria. Some bacteria even have cell membrane rupture and leakage of contents.

[0017] In a seventh aspect of the invention, an antibacterial pharmaceutical composition is provided, the composition comprising the polypeptide and a pharmaceutically acceptable carrier.

[0018] Furthermore, the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, or colorants.

[0019] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, or dispersants.

[0020] Furthermore, the broad-spectrum antibacterial activity and bactericidal mechanism of the defensin of *Ixodes persulcatus* were investigated.

[0021] Furthermore, the bacterial strain is one of Staphylococcus aureus (S. aureus CCTCC AB94004, S. aureus CCTCC AB 204036), Staphylococcus epidermidis (S. epidermidis CCTCC AB208187, S. epidermidis CCTCC AB208188) and Escherichia coli (E. coli ATCC8739, E. coli ATCC25922).

[0022] Preferably, the defensin IpDf3 derived from *Ixodes persulcatus* exhibits broad-spectrum resistance to both Gram-positive and Gram-negative bacteria, and its bactericidal mechanism primarily involves direct cell membrane disruption.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention annotated a novel defensin peptide, IpDf3, from the *Ixodes persulcatus* genome database and successfully prepared the peptide using affinity chromatography, EK restriction enzyme digestion, RP-HPLC, and mass spectrometry. This peptide exhibits broad-spectrum antibacterial activity due to its unique structure (containing three pairs of intramolecular disulfide bonds formed by six cysteine ​​residues, with pairing arrangements of Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6), making it suitable for drug development against both Gram-positive and Gram-negative bacteria. Its core advantages include: (1) Ultra-high antibacterial activity: The minimum inhibitory concentration experiment showed that the recombinant whole-sulcus tick defensin Ox-IpDf3 had different degrees of antibacterial activity against four Gram-positive bacteria and two Gram-negative bacteria. In particular, the MIC was the lowest against S. aureus AB94004 and S. epidermidis AB208188, which was about 0.0625 μM, which was superior to traditional antibiotics such as vancomycin.

[0024] (2) Rapid bactericidal mechanism: By directly destroying the bacterial cell membrane, it causes the membrane surface to become concave within 30 minutes and the membrane to rupture and leak contents within 2 hours, effectively avoiding the development of drug resistance. Bactericidal kinetics and scanning electron microscopy experiments showed that 3×MIC IpDf3 rapidly downregulated the colony count by 1-4 log units within 60 minutes, and all of them caused irregular protrusions or depressions on the surface of Gram-positive and Gram-negative bacteria, and even cell membrane rupture, indicating that its antibacterial mechanism may be closely related to direct damage to the cell membrane.

[0025] (3) Wide range of applications: This invention has discovered a novel antimicrobial defensin IpDf3 derived from the tick tick. It not only has the activity of traditional defensins against Gram-positive bacteria, but also has a certain antimicrobial activity against Gram-negative bacteria. It has a broad antimicrobial spectrum, strong activity, and fast bactericidal speed. It can be prepared into anti-infective drugs (such as injections and sprays), medical device coatings, or anti-biofilm preparations. In particular, it is expected to provide candidates for the development of new antimicrobial drugs and has important application prospects for drug-resistant bacterial infections caused by ESKAPE pathogens. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Gene annotation of the defensin IpDf3 from *Ixodes persulcatus*.

[0028] Figure 2 Specific structural information on defensin peptides derived from *Ixodes persulcatus*.

[0029] Figure 3Expression, purification, and identification of recombinant peptide IpDf3: (A) Amino acid sequence information of IpDf3, a defensin from *Ixodes sulcus*. (B) SDS-PAGE analysis of IpDf3 expressed and purified in *Escherichia coli* BL21(DE3). Lane 1, uninduced cell-free extract of *E. coli* containing pET-32a-IpDf3; Lane 2, total cell-free extract induced by IPTG; Lane 3, sonicated supernatant from induced *E. coli*; Lane 4, sonicated deposits from induced *E. coli*; Lane 5, His fusion protein purified by affinity chromatography; Lane 6, fusion protein cleaved by enterokinase; IpDf3 purified by HPLC, M representing the protein label. (C) HPLC purity analysis of IpDf3 using analytical columns. (D) MALDI-TOF-MS mass spectrometry of purified IpDf3. (E) 3D structural model of IpDf3 using BmKDfsin3 as a homology model.

[0030] Figure 4 The images show the inhibition zones produced by treatment with peptides Re-IpDf3 and Ox-IpDf3. (A) These images represent different antimicrobial treatments for Staphylococcus aureus ATCC204036. (B) These images represent different antimicrobial treatments for Staphylococcus aureus AB94004. (C) These images represent different antimicrobial treatments for Staphylococcus epidermidis AB208187. (D) These images represent different antimicrobial treatments for Staphylococcus epidermidis AB208188. (E) These images represent different antimicrobial treatments for Escherichia coli ATCC8739. (F) These images represent different antimicrobial treatments for Escherichia coli ATCC25922. For each plate, the first row from left to right represents Ox-IpDf3 and Re-IpDf3, and the second row from left to right represents the negative and positive controls.

[0031] Figure 5 To investigate the effect of DTT treatment on the antibacterial activity of Ox-IpDf3, (A) the effect of 5 mM DTT on the growth of four bacteria. (B) the effect of DTT treatment on the antibacterial activity of Ox-IpDf3.

[0032] Figure 6 The effect of cysteine ​​mutations on the antibacterial activity of IpDf3. (A) Amino acid sequence alignment of the three IpDf3 mutations. (B) HPLC purity analysis of IpDf3 (C4-A). (C) HPLC purity analysis of IpDf3 (C11-A). (D) HPLC purity analysis of IpDf3 (C15-A). (E) MIC of the three IpDf3 mutations on bacterial strains.

[0033] Figure 7This study investigates the bactericidal kinetics and mechanism of action of the defensin IpDf3 from *Ixodes persulcatus*. A represents the bactericidal kinetics, and B represents morphological evidence of destruction obtained via scanning electron microscopy (SEM). Detailed Implementation

[0034] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0035] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0036] Unless otherwise specified, all drugs, reagents or instruments used in this invention are commercially available.

[0037] The broad-spectrum antibacterial activity and bactericidal mechanism of the whole-sulcus tick defensin IpDf3 of this application will be described in detail below with reference to embodiments and experimental data.

[0038] Example 1: Gene annotation of the defensin IpDf3 in *Ixodes persulcatus* A search for "Ixodes persulcatus defensin" in the NCBI database yielded one documented defensin from *Ixodes persulcatus* (GenBank accession number: BAH09304.1). The nucleic acid and protein sequences of this defensin were downloaded. Next, using this *Persulcatusin* sequence as a reference, a search for defensins from *Ixodes persulcatus* using the keyword "tblastn" was conducted. Thirteen highly similar homologous sequences were obtained, named IpDf1-IpDf13. Their protein sequences were downloaded and saved in FASTA format. Using a 2000 bp range upstream and downstream of the nucleic acid sequence corresponding to IpDf3 as a search area, the open reading frame (ORF) was determined by identifying the start and stop codons. The obtained nucleic acid sequence was then compared with the known CDS sequence of Persulcatusin, and the splicing mode of the exon was determined according to the GT-AG rule. Then, the signal peptide was predicted using the SignaIP 5.0 website to annotate the gene structure of the persulcatusin IpDf3.

[0039] The results are as follows Figure 1As shown, IpDf3 contains three exons and two introns. The first intron is short, with 82 bp, and splits the signal peptide in two; the second intron is located at the end of the propeptide and is 1684 bp in length. After cleavage, the complete whole-sulcus tick defensin precursor protein is formed.

[0040] Example 2: Prokaryotic expression and identification of the defensin IpDf3 gene from *Ixodes persulcatus* Based on the amino acid sequence of the defensin IpDf3, the corresponding nucleotide sequence was obtained through reverse translation and then codon-optimized (https: / / www.novopro.cn / tools / codon-optimization.html) to ensure efficient and high-volume expression in E. coli. The codon-optimized nucleotide sequence is shown in SEQ ID NO.2.

[0041] Next, primer fragments were designed using the overlap-PCR method (primers are shown in Table 1) to contain recombinant enterokinase cleavage site 'DDDDK', stop codon TAG, Kpn I cleavage site, Xho I cleavage site, and protective bases, so that they could be correctly inserted into the expression vector pET-32a (commercially available, such as UBO Bio #VT1216).

[0042] Table 1

[0043] Using molecular biology techniques such as PCR amplification, enzyme digestion, ligation, transformation, plasmid extraction, and DNA sequencing, the recombinant expression plasmid pET-32a-IpDf3 was successfully obtained. The correctly sequenced recombinant plasmid was transformed into the expression strain *E. coli* BL21(DE3), and the expression conditions of the HIS-IpDf3 fusion protein were optimized by changing the concentration of IPTG, temperature, and induction time. Based on the optimized induction conditions, the fusion protein was isolated and purified using methods such as ultrasonic disruption, centrifugation, affinity chromatography, ultrafiltration, enzyme digestion, and RP-HPLC, successfully preparing the defensin IpDf3 from *Ixodes persulcatus*. Finally, the purity of the recombinant defensin IpDf3 was determined using analytical reversed-phase high-performance liquid chromatography (RP-HPLC), and the relative molecular mass of the recombinant defensin peptide from *Ixodes persulcatus* was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0044] SDS-PAGE electrophoresis results are as follows: Figure 3As shown, bacterial protein levels significantly increased after IPTG induction, and the protein bands after EK digestion and purification ranged from 3.3 kDa to 6.5 kDa, consistent with the theoretical value of 4.2 kDa, indicating successful expression of the defensin peptide IpDf3. Subsequently, the defensin IpDf3 from *Ixodes sulci* was purified by RP-HPLC semi-preparative column separation and vacuum freeze-drying, successfully yielding chromatographically pure IpDf3 peptide. Further analytical RP-HPLC was used to determine its purity. The results showed that the recombinant defensin IpDf3 exhibited a single target peak at 18.3 min (…). Figure 3 C) indicates that the prepared recombinant whole-sulcus tick defensin IpDf3 has high purity. MALDI-TOF-MS mass spectrometry identification shows that the molecular weight of the recombinant expressed peptide IpDf3 is 4211.20 Da, which is about 6 Da less than the theoretical molecular weight (4217.87 Da). Figure 3 D). This is because its molecule contains 6 cysteine ​​residues, which can form 3 pairs of disulfide bonds during prokaryotic expression, thereby removing 6 hydrogen atoms, consistent with theoretical values. Three-dimensional structure prediction results show that the whole-sulcus tick defensin IpDf3 contains one α-helix and two β-sheets, with 6 cysteine ​​residues forming 3 pairs of disulfide bonds inside the molecule, paired in Cys1-Cys4, Csy2-Cys5, and Cys3-Cys6 configurations, consistent with expectations. Figure 3 E).

[0045] In summary, the oxidized IpDf3 (Ox-IpDf3) peptide was successfully prepared through prokaryotic expression, providing experimental materials for subsequent functional studies.

[0046] Example 3: Inhibitory activity of the defensin IpDf3 from *Ixodes persulcatus* against Gram-negative bacteria. I. Determination of Minimum Inhibitory Concentration (MIC) Six bacterial strains (see Table 1) were inoculated into MHB broth medium and cultured at 37 °C and 200 rpm until the logarithmic growth phase (OD200). 630 (Approximately 0.25), continue to dilute 100 times with MHB liquid medium until the bacterial concentration is 1×10⁻⁵. 5CFU / mL, and 100 μL of bacterial culture was added to a 96-well plate. Next, 100 μL of bacterial culture and 100 μL of oxidized IpDf3 peptide (Ox-IpDf3) solution were added to the 96-well plates to achieve final peptide concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μM. The negative control group consisted of 200 μL of MHB medium, and the positive control group consisted of 100 μL of MHB medium and 100 μL of bacterial culture. Antibiotic control groups for vancomycin and kanamycin were also included, with three replicates for each sample concentration. After incubation at 37 ℃ and 100 rpm for 12–16 h, the absorbance of the samples at 630 nm was measured using a multi-mode microplate reader. The minimum inhibitory concentration (MIC) of defensin IpDf3 was defined as the lowest concentration at which bacterial cell precipitation was not visible to the naked eye. The table below summarizes the MIC values ​​of recombinant whole-sulcus tick defensin Ox-IpDf3 against four Gram-positive and two Gram-negative standard bacterial strains.

[0047] Table 2

[0048] Note: "Kan" represents kanamycin, and "Van" represents vancomycin. As shown in Table 1: 1. Susceptibility to Gram-positive bacteria Ox-IpDf3 exhibited extremely high antibacterial activity against four Gram-positive bacteria (MIC 0.0625-0.25 μM). It exhibits the strongest activity against Staphylococcus aureus AB94004 and Staphylococcus epidermidis AB208188 (MIC=0.0625 μM). Its activity was significantly better than that of vancomycin (8-32 times) and kanamycin (64-256 times).

[0049] 2. Inhibition of Gram-negative bacteria The MIC value for the two Escherichia coli strains (ATCC25922 / ATCC8739) was 8 μM, which was more than twice as effective as kanamycin, while vancomycin was basically ineffective against Gram-negative bacteria (MIC>2 μM).

[0050] Example 4, Mechanism Study 1. Differences in antibacterial activity between reduced and oxidized IpDf3 To further investigate the effect of the structure of the defensin IpDf3 from *Ixodes persulcatus* on its antibacterial activity, the effects of chemically synthesized 100 μM reduced IpDf3 (Re-IpDf3) and recombinantly expressed oxidized IpDf3 polypeptide (Ox-IpDf3) solutions on the growth of six standard strains were compared and analyzed using the inhibition zone experiment.

[0051] The results show that ( Figure 4 In all six plates (A, B, C, D, E, F), a clear inhibition zone appeared around the tablets treated with oxidized Ox-IpDf3, while none appeared around the tablets treated with reduced Re-IpDf3. This indicates that the presence or absence of disulfide bonds in IpDf3 is crucial to its antibacterial activity. Furthermore, the inhibition zone effect of Ox-IpDf3 was more pronounced on Gram-positive bacteria plates than on Gram-negative bacteria plates, further demonstrating that the defensin IpDf3 has a stronger inhibitory effect on the four Gram-positive bacteria than on the two Gram-negative bacteria, consistent with the experimental conclusions regarding the minimum inhibitory concentration.

[0052] 2. Effects of DTT treatment on the antibacterial function of IpDf3 To further verify the effect of disulfide bonds on the antibacterial function of IpDf3, the peptide was treated with the reducing agent dithiothreitol (DTT). Neither 1 mM nor 2 mM DTT could open all disulfide bonds, and 10 mM DTT was toxic to cells. Therefore, the effect of 5 mM DTT on the growth of four Gram-positive bacteria was first determined.

[0053] The results showed that ( Figure 5 A) Compared with the control group, 5 mM DTT had almost no effect on the growth of the four strains. This concentration of DTT can be used to treat the peptide, causing the oxidized Ox-IpDf3 peptide to unfold into a linear structure. Then, the effects of DTT-treated Ox-IpDf3 and untreated Ox-IpDf3 on the growth of two Staphylococcus aureus strains were measured. S. aureus AB94004, ATCC204036) and two types of Staphylococcus epidermidis ( S. epidermidis The antibacterial activity of AB208187 and AB208188 was observed. The results showed (…). Figure 5 B), compared with the Ox-IpDf3 treatment group, the DTT-treated Ox-IpDf3 lost its antibacterial activity at different concentrations, indicating that the disulfide bonds in the oxidized Ox-IpDf3 peptide are very important for its antibacterial activity.

[0054] 3. Effect of cysteine ​​mutation on the antibacterial activity of IpDf3 Site-directed mutagenesis of cysteine ​​residues in polypeptide molecules can directly disrupt disulfide bond structures. Using chemical synthesis and random oxidation methods, three mutant polypeptides of IpDf3 (C4-A, C11-A, and C15-A) were obtained. Figure 6 A). RP-HPLC results showed that all three mutant peptides exhibited a single absorption peak, indicating high purity of the synthesized peptides. Furthermore, due to changes in sequence structure, the elution peak retention times of the three mutants shifted compared to IpDf3 (18.3 min), specifically, the elution time for mutant IpDf3 (C4-A) was 20.4 min (Fig. 5B), and the elution time for IpDf3 (C11-A) was 19.2 min (Fig. 5B). Figure 6 The elution time for C), IpDf3 (C15-A) was 18.7 min ( Figure 6 D).

[0055] Antibacterial experiments showed that with the disruption of disulfide bonds between peptide molecules, the antibacterial activity of the three IpDf3 mutant peptides against *S. aureus* AB94004, *S. epidermidis* AB208188, and *E. coli* ATCC25922 was significantly reduced, especially against the two Gram-positive bacteria, where the MIC values ​​increased to 8-16 μM. Figure 6 E). These results further demonstrate that disulfide bonds can affect the antibacterial bioactivity of IpDf3.

[0056] Example 4: Bactericidal kinetics and mechanism of action of the whole-sulcus tick defensin IpDf3 against Gram-negative bacteria. Dilute the revived bacterial culture 100 times with fresh MHB liquid medium to achieve a concentration of 10. 7 The bacterial culture was approximately CFU / mL. Next, the bacterial culture was treated with IpDf3 at final concentrations of 3× and 5× MIC, respectively, and a positive control group (vancomycin / kanamycin) and a negative control group (0.9% physiological saline) were set up. Then, at five time points (0 min, 5 min, 15 min, 30 min, and 60 min), 200 μL of each group of samples were taken, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the samples were resuspended in 200 μL of sterile physiological saline. After six 10-fold serial dilutions, 10 μL of each group of bacterial culture was evenly spread on LB agar plates and incubated overnight at 37 ℃. Finally, plates with appropriate colony counts (40-200 CFU / mL) were selected for colony counting, and the corresponding bacterial concentrations were calculated according to the dilution factor. The statistical results were then analyzed using GraphPad and curves were plotted.

[0057] The changes in bacterial membrane morphology after treatment with defensin from *Ixodes persulcatus* were observed using scanning electron microscopy (SEM) to further investigate the bactericidal mechanism of defensin. The sample preparation steps for SEM are as follows: (1) Sampling: 1 mL of bacterial culture was cultured until OD630 was 0.25, and 3×MIC of polypeptide IpDf3 was added and incubated for 30 min, 1 h and 2 h respectively. Then the culture medium was discarded by centrifugation and the bacterial cells were washed with physiological saline 3-5 times. (2) Fixation: Resuspend the bacterial culture in 500 μL of 2.5% glutaraldehyde fixative, spread the bacterial culture on a coverslip and dry at 37 °C for 2 h, then fix overnight with fresh 2.5% glutaraldehyde (diluted with 100 mM PBS pH 7.4); (3) Washing: Remove the fixative and wash with PBS 3 times, each time for 15 min; (4) Gradient dehydration: 30%, 50%, 70%, 80%, 90%, 100% ethanol gradient dehydration, 15 min per grade, 100% ethanol repeated once; (5) Freeze-drying: The treated material is placed in a basket and then placed in a carbon dioxide critical point dryer; (6) Sputtering: The dried sample is attached to the sample stage with conductive adhesive and then placed in an ion sputtering instrument for sputtering. (7) Observe and photograph using a scanning electron microscope. The results are as follows: Figure 7 As shown.

[0058] Depend on Figure 7 As shown in Figure A, compared to the negative control group, the colony count in the IpDf3 treatment groups at different concentrations decreased significantly over time, and the 5×MIC concentration of IpDf3 exhibited a faster bactericidal rate against S. epidermidis AB208188 within 15 min. Anions exist in the bacterial plasma membrane; amphiphilic defensins carrying cationic ions combine with the negatively charged bacterial surface, subsequently inserting into the hydrophobic region of the lipid membrane, thus causing membrane damage and decomposition.

[0059] Depend on Figure 7 As shown in Figure B, after co-culturing bacteria with the IpDf3 peptide for 30 min, the surface of some bacteria shrank and became irregular; after co-culturing for 2 h, the morphological changes were severe, with irregular protrusions and depressions appearing on the bacterial surface, and some bacteria exhibited cell membrane rupture and leakage of contents, indicating that it has a direct bactericidal effect. In summary, the defensin IpDf3 from *Ixodes persulcatus* may exert its antibacterial properties through direct membrane disruption.

[0060] In summary, this invention has practical value. (1) Broad spectrum coverage: It can kill Gram-positive bacteria (such as Se 208188) and Gram-negative bacteria (such as Ec25922) simultaneously and effectively.

[0061] (2) Clinical potential: It can overcome the outer membrane barrier of Gram-negative bacteria (such as MIC 0.0625μM, which is superior to vancomycin) and drug-resistant Gram-positive bacteria (which is difficult to achieve with traditional defensins).

[0062] (3) Development direction: Infected wound spray (rapidly clear complex bacterial groups), duct coating (prevent biofilm formation).

[0063] Finally, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0064] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A defensin polypeptide derived from *Ixodes sulci*, characterized in that, The defensin polypeptide is IpDf3, and its amino acid sequence is shown in SEQ ID NO: 1; The defensin polypeptide IpDf3 contains an α-helix, two β-sheets, and three pairs of disulfide bonds formed by six cysteine ​​residues, paired in the manner of Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6.

2. A method for high-yield of the defensin polypeptide of claim 1, characterized in that, Includes the following steps: The amino acid sequence of the codon-optimized defensin peptide IpDf3 was inserted into the expression vector pET-32a to obtain a recombinant expression plasmid. The recombinant plasmid was then transformed into E. coli BL21(DE3) to obtain a genetically engineered E. coli strain that produces a high amount of defensin peptide IpDf3. The optimized nucleotide sequence of the codon is shown in SEQ ID NO:

2.

3. The use of the polypeptide of claim 1 in the preparation of a drug for treating Gram-positive and / or Gram-negative bacteria; characterized in that, The Gram-positive bacteria is Staphylococcus aureus; the Gram-negative bacteria is at least one of Escherichia coli ATCC8739 and Escherichia coli ATCC25922.

4. An antibacterial drug composition, characterized in that, The composition comprises the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

5. The antibacterial drug composition according to claim 4, characterized in that, The dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, or dispersants.

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