Novel defensin polypeptide derived from sclerophyllum squarrosum and application of novel defensin polypeptide

Abstract

The invention discloses a novel defensin polypeptide sourced from sclerophyllum squarrosum and application of the novel defensin polypeptide. The novel defensin polypeptide is IpDf3, and the amino acid sequence of the novel defensin polypeptide is shown as SEQ ID NO: 1. The compound has different degrees of antibacterial activity to four gram-positive bacteria and two gram-negative bacteria. Bactericidal kinetics and scanning electron microscope experiments show that the IpDf3 of 3 * MIC rapidly down-regulates the colony count by 1-4 logarithmic units within 60 min, and can cause irregular protrusions or recesses on the surfaces of gram-positive bacteria and even rupture of cell membranes, which indicates that the antibacterial mechanism of the IpDf3 is possibly closely related to direct damage of the cell membranes. The defensin polypeptide not only has the anti-gram-positive bacterium activity of traditional defensin, but also has certain antibacterial activity on gram-negative bacteria, is wide in antibacterial spectrum, high in activity and high in sterilization speed, is expected to provide candidates for research and development of novel antibacterial drugs, and has important application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to the discovery of a new type of defense peptide IpDf3 from Ixodes persulcatus and the research on its antibacterial activity and mechanism. BACKGROUND

[0002] As a global distribution of obligate hematophagous ectoparasite, tick is the second vector after mosquito. It can transmit a variety of pathogens through biting or blood sucking, which seriously threatens human health and the development of animal husbandry. However, it can be immune to infection in the process of contacting a large number of pathogens, which implies that it may be rich in special active substances to defend pathogens in the body. Defensin is a kind of cationic polypeptide rich in disulfide bond, which is widely distributed in fungi, plants, mammals, scorpions, mollusks and ticks, and is an important component of the innate immune system of organisms. It has inhibitory activity to bacteria, viruses and fungi and other microorganisms, and has high research value and wide application prospect. Due to its unique antibacterial mechanism, defensin has become an important molecular skeleton for the development of new antibacterial drugs. At the same time, bacterial resistance is a global public health problem, and most antibiotic-resistant infections are caused by ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter). There are a variety of insects, which contain rich defensin resources. Its unique polypeptide structure and natural broad-spectrum antibacterial activity are expected to provide new strategies for the research and development of new antibacterial drugs. Therefore, it is urgent to develop new antibacterial drugs that can replace antibiotics, especially natural active substances from the body of insects. SUMMARY

[0003] The purpose of the present application is to discover a new antibacterial defensin IpDf3 from Ixodes persulcatus and its bactericidal mechanism, which not only has the activity of traditional defensin against gram-positive bacteria, but also has certain antibacterial activity against gram-negative bacteria. It 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 prospect. The present application adopts the following technical scheme: In the first aspect of the present application, a new type of defensin polypeptide from Ixodes persulcatus is provided, and the new type of defensin polypeptide is IpDf3, and the amino acid sequence is shown as SEQ ID NO: 1.

[0004] Further, the specific structural information is as follows: Figure 2The amino acid sequence of the reduced form IpDf3 is shown in SEQ ID NO: 1. The reduced form IpDf3 is obtained by chemical synthesis, and the oxidized form IpDf3 polypeptide (Ox-IpDf3) is obtained by in vitro expression, which contains 3 pairs of intramolecular disulfide bonds formed by 6 cysteines, and the pairing mode is Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6.

[0005] In a second aspect of the present application, a nucleic acid molecule encoding the polypeptide is provided.

[0006] In a third aspect of the present application, a recombinant expression vector containing the nucleic acid molecule is provided.

[0007] In a fourth aspect of the present application, a recombinant bacterium or an engineered host cell line comprising the recombinant expression vector is provided.

[0008] In a fifth aspect of the present application, a preparation method of the defensin polypeptide is provided, which comprises: cloning a gene fragment encoding the novel defensin polypeptide IpDf3 into a vector to obtain a recombinant vector; inducing expression of the recombinant vector, and purifying the defensin polypeptide.

[0009] As another embodiment, the defensin polypeptide can also be directly synthesized according to the amino acid sequence.

[0010] In a sixth aspect of the present application, the polypeptide is used in the preparation of an anti-Gram-positive and / or Gram-negative bacteria drug.

[0011] Further, the Ixodes vespertilionis defensin IpDf3 has different degrees of antibacterial activity against 4 kinds of Gram-positive bacteria and 2 kinds of Gram-negative bacteria.

[0012] The Gram-positive bacteria include at least one of Staphylococcus aureus and Staphylococcus epidermidis.

[0013] The Gram-negative bacteria include at least one of E. coli ATCC8739 and E. coli ATCC25922. Further, the Ixodes vespertilionis defensin IpDf3 has the lowest MIC of about 0.0625 μM against the Gram-positive bacteria S. aureus AB94004 and S. epidermidis AB208188, and also shows certain antibacterial activity against E. coli ATCC25922 and E. coli ATCC8739, with the MIC value of about 8 μM.

[0014] Further, the IpDf3 at 3xMIC can rapidly down-regulate the bacterial colony number by 1-4 logarithmic units in 60 min. Further, the IpDf3 at 3xMIC can rapidly down-regulate the bacterial colony number by 1-4 logarithmic units in 60 min.

[0015] Further, the defensin IpDf3 can cause irregular protuberances or depressions on the surface of gram-positive and gram-negative bacteria at 30 min, and after 2 h of treatment, the morphological changes are severe, irregular protuberances and depressions appear on the surface of bacteria, and individual bacteria have phenomena of cell membrane rupture and content leakage.

[0016] In a seventh aspect of the present application, an antibacterial pharmaceutical composition is provided, which comprises the polypeptide and a pharmaceutically acceptable carrier.

[0017] Further, the excipient comprises at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

[0018] Further, the dosage form of the medicine comprises at least one of granules, tablets, pills, capsules, injections or dispersants.

[0019] Further, the broad-spectrum antibacterial activity and bactericidal mechanism of the Ixodes persulcatus defensin.

[0020] Further, 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).

[0021] Preferably, the Ixodes persulcatus-derived defensin IpDf3 has broad-spectrum resistance to gram-positive and gram-negative bacteria, and a direct bactericidal mechanism mainly by destroying the cell membrane.

[0022] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: The present application annotates a novel defensin polypeptide IpDf3 from the genome database of Ixodes persulcatus, and successfully prepares the polypeptide by using methods such as affinity chromatography, EK enzyme digestion, RP-HPLC high performance liquid chromatography and mass spectrometry identification. The polypeptide realizes broad-spectrum antibacterial activity through its unique structure (containing 3 pairs of intramolecular disulfide bonds formed by 6 cysteines, the pairing mode is Cys1-Cys4, Cys2-Cys5, Cys3-Cys6), and is suitable for the development of drugs against gram-positive bacteria and gram-negative bacteria, and its core advantages include: (1) Ultra-high antibacterial activity: The minimum inhibitory concentration experiment shows that the recombinant full-groove hard tick defense factor Ox-IpDf3 has antibacterial activity of different degrees on 4 kinds of gram-positive bacteria and 2 kinds of gram-negative bacteria, especially the MIC of S. aureus AB94004 and S. epidermidis AB208188 is the lowest, about 0.0625 μM, which is better than traditional antibiotics such as vancomycin.

[0023] (2) Rapid bactericidal mechanism: by directly destroying the bacterial cell membrane, causing the membrane surface to be concave within 30 minutes, and causing the membrane to rupture and the contents to leak within 2 hours, effectively avoiding the generation of drug resistance. Bactericidal kinetics and scanning electron microscope experiments show that 3xMIC IpDf3 rapidly down-regulates the number of bacteria by 1-4 logarithmic units within 60 min, and can cause irregular bulges or concave on the surface of gram-positive bacteria and gram-negative bacteria, and even rupture of the cell membrane, indicating that its antibacterial mechanism may be closely related to the direct damage of the cell membrane.

[0024] (3) Wide application scenarios: the present application finds a new antibacterial defense factor IpDf3 from full-groove hard tick, which not only has the activity of traditional defense factor against gram-positive bacteria, but also has certain antibacterial activity against gram-negative bacteria, wide antibacterial spectrum, strong activity, fast bactericidal speed, can be prepared into anti-infection drugs (such as injection, spray), medical device coating or antibiofilm preparation, especially for drug-resistant bacterial infection caused by ESKAPE pathogens, which is expected to provide candidates for the research and development of new antibacterial drugs, and has important application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0026] Figure 1 Gene annotation of full-groove hard tick-derived defense factor IpDf3.

[0027] Figure 2 Specific structural information of the novel defense factor polypeptide derived from full-groove hard tick.

[0028] Figure 3Figure 1. Expression, purification and characterization of recombinant peptide IpDf3 (A) Amino acid sequence information of IpDf3, a hard tick gut defensin. (B) SDS-PAGE analysis of IpDf3 expressed and purified in E. 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, supernatant of ultrasonic lysate from induced E. coli; Lane 4, sediment of ultrasonic lysate from induced E. coli; Lane 5, purified His fusion protein after affinity chromatography; Lane 6, fusion protein after cleavage by enterokinase; IpDf3 after HPLC purification, M represents protein marker. (C) HPLC purity analysis of IpDf3 using analytical column. (D) MALDI-TOF-MS mass spectrum of purified IpDf3. (E) 3D structure model of IpDf3 using BmKDfsin3 as homology model.

[0029] Figure 4 Figure 4. Inhibition zones generated by peptide Re-IpDf3 and Ox-IpDf3 treatments, (A) These images represent different antibacterial treatments of S. aureus ATCC204036. (B) These images represent different antibacterial treatments of S. aureus AB94004. (C) These images represent different antibacterial treatments of S. epidermidis AB208187. (D) These images represent different antibacterial treatments of S. epidermidis AB208188. (E) These images represent different antibacterial treatments of E. coli ATCC8739. (F) These images represent different antibacterial treatments of E. coli ATCC25922. For each plate, the first row from left to right is Ox-IpDf3 and Re-IpDf3, the second row from left to right is negative control and positive control.

[0030] Figure 5 Figure 5. DTT treatment affects antibacterial activity of Ox-IpDf3, (A) Effect of 5 mM DTT on the growth of four bacteria. (B) Effect of DTT treatment on antibacterial activity of Ox-IpDf3.

[0031] Figure 6 Figure 6. Effect of cysteine mutations on antibacterial activity of IpDf3, (A) Alignment of amino acid sequences of three IpDf3 mutants. (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 three IpDf3 mutants on bacterial strains.

[0032] Figure 7To study the bactericidal kinetics and mechanism of Ixodes persulcatus defensin IpDf3. A is the bactericidal kinetics, B is the morphological damage evidence of scanning electron microscopy (SEM). DETAILED DESCRIPTION

[0033] The advantages and various effects of the present application will be more clearly presented hereinafter with specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0034] Throughout this specification, unless otherwise specifically indicated otherwise, the terms used herein are understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. If there is a contradiction, the present specification takes precedence.

[0035] Unless otherwise specifically stated, the drugs, reagents or instruments used in the present application can be purchased from the market.

[0036] The broad-spectrum antibacterial activity and bactericidal mechanism of Ixodes persulcatus defensin IpDf3 will be described in detail below with examples and experimental data.

[0037] Example 1: Gene annotation of Ixodes persulcatus defensin IpDf3 In the NCBI database, search for "Ixodes persulcatus defensin" to get one recorded Ixodes persulcatus defensin Persulcatusin (GenBank accession number: BAH09304.1), download the nucleic acid sequence and protein sequence of the defensin. Then, taking the Persulcatusin sequence as a reference, search for Ixodes persulcatus defensin in the Ixodes persulcatus species "tblastn" to get 13 similar high homologous sequences, name these sequences IpDf1-IpDf13, download their protein sequences and save them as fasta format. Taking the upstream and downstream 2000 bp of the nucleic acid sequence corresponding to IpDf3 as the search range, determine its open reading frame (ORF) by searching for the start codon and stop codon. Then compare the obtained nucleic acid sequence with the known Persulcatusin CDS sequence, and determine the splicing mode of the exons according to the GT-AG rule, then use the SignaIP 5.0 website to predict the signal peptide, and annotate the gene structure of Ixodes persulcatus defensin IpDf3.

[0038] Results are as follows Figure 1As shown, IpDf3 contains three exons and two introns. The first intron is shorter, with a base number of 82 bp, which divides the signal peptide into two parts; the second intron is located at the end of the propeptide, with a length of 1684 bp. After splicing, the complete full-groove tick defense peptide precursor protein is formed.

[0039] Example 2, Prokaryotic expression and identification of full-groove tick defense peptide IpDf3 gene According to the amino acid sequence of the defense peptide IpDf3, the corresponding nucleotide sequence is obtained by reverse translation, and codon optimization is performed (https: / / www.novopro.cn / tools / codon-optimization.html) to ensure efficient and large-scale expression in E. coli. The codon-optimized nucleotide sequence is shown as SEQ ID NO. 2.

[0040] Next, the primer fragments (see Table 1) are designed using the Overlap-PCR method, which contains the recombinant enterokinase cleavage site 'DDDDK', the stop codon TAG, the Kpn I enzyme cleavage site, the Xho I enzyme cleavage site and the protection base, etc., so as to be correctly inserted into the expression vector pET-32a (commercially available, such as Novoprotein #VT1216).

[0041] Table 1

[0042] By means of PCR amplification, enzyme digestion, ligation, transformation, plasmid extraction and DNA sequencing, etc. molecular biology techniques, the pET-32a-IpDf3 recombinant expression plasmid is successfully obtained. The recombinant plasmid with correct sequencing is transformed into the expression strain E. coli BL21 (DE3), and the conditions for expression of HIS-IpDf3 fusion protein are optimized by changing the concentration of IPTG, temperature and induction time. According to the optimized induction expression conditions, the fusion protein is separated and purified by means of ultrasonic crushing, centrifugation, affinity chromatography, ultrafiltration, enzyme digestion and RP-HPLC, etc. The full-groove tick defense peptide IpDf3 is successfully prepared. Finally, the purity of the recombinant defense peptide IpDf3 is detected by using analytical reverse-phase high-performance liquid chromatography RP-HPLC, and the relative molecular mass of the recombinant full-groove tick defense peptide polypeptide is determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0043] The 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).

[0044] In summary, the oxidized IpDf3 (Ox-IpDf3) peptide was successfully prepared through prokaryotic expression, providing experimental materials for subsequent functional studies.

[0045] 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 the bacterial solution was taken in a 96-well culture plate. Then, 100 μL of the bacterial solution and 100 μL of an oxidized IpDf3 polypeptide (Ox-IpDf3) solution were added to the 96-well culture plate, so that the final concentration of the polypeptide in the well was 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μM. The negative control group was 200 μL of MHB medium, the positive control group was 100 μL of MHB medium and 100 μL of the bacterial solution, and antibiotic control groups of vancomycin and kanamycin were also set up, and three groups of parallel repeats were set up for each sample concentration. After incubation in a constant-temperature shaker at 37 °C and 100 rpm for 12-16 h, the absorbance value of the sample at 630 nm was detected by a multifunctional enzyme label instrument. The minimum antibacterial peptide concentration at which no bacterial body precipitate could be seen with the naked eye was the minimum inhibitory concentration (MIC) of the defensin IpDf3. The following table summarizes the MIC values of the recombinant full-groove hard tick defensin Ox-IpDf3 against four gram-positive bacteria and two gram-negative bacteria standard strains.

[0046] Table 2

[0047] Note: "Kan" represents kanamycin, and "Van" represents vancomycin From Table 1, it can be seen that: 1. Gram-positive bacteria sensitivity Ox-IpDf3 showed super-high antibacterial activity (MIC 0.0625-0.25 μM) against four gram-positive bacteria; The activity against Staphylococcus aureus AB94004 and Staphylococcus epidermidis AB208188 was the strongest (MIC = 0.0625 μM); The activity was significantly better than that of vancomycin (8-32 times) and kanamycin (64-256 times).

[0048] 2. Gram-negative bacteria inhibition The MIC value against two Escherichia coli strains (ATCC25922 / ATCC8739) was 8 μM, and the activity was better than that of kanamycin (more than 2 times), while vancomycin was basically ineffective against gram-negative bacteria (MIC > 2 μM).

[0049] Example 4, mechanism research 1. Difference in antibacterial activity of reduced and oxidized IpDf3 To further explore the influence of the structure of I. persulcatus defensin IpDf3 on its antibacterial activity, the effects of 100 μM reduced IpDf3 (Re-IpDf3) and oxidized IpDf3 polypeptide (Ox-IpDf3) on the growth of 6 standard strains were compared and analyzed by means of inhibition zone experiment.

[0050] The results showed that Figure 4 A, B, C, D, E, F), obvious inhibition zones appeared around the tablets treated with Ox-IpDf3 in 6 plates, while no inhibition zone appeared around the tablets treated with Re-IpDf3, indicating that the presence or absence of disulfide bond in IpDf3 is crucial for its antibacterial activity. In addition, the antibacterial effect of Ox-IpDf3 on gram-positive bacteria plates was relatively more obvious than that on gram-negative bacteria plates, which again indicated that the inhibitory effect of defensin IpDf3 on 4 gram-positive bacteria was stronger than that on 2 gram-negative bacteria, which was consistent with the conclusion of the minimum inhibitory concentration experiment.

[0051] 2, Effect of DTT treatment on antibacterial function of IpDf3 To further verify the influence of disulfide bond on the antibacterial function of IpDf3, the polypeptide was treated with reducing agent dithiothreitol (DTT). 1 mM and 2 mM DTT cannot open all disulfide bonds, and 10 mM DTT has certain toxicity to cells, so the effect of 5 mM DTT on the growth of 4 gram-positive bacteria was first determined.

[0052] The results showed that Figure 5 A), compared with the control group, 5 mM DTT had little effect on the growth of 4 strains, and DTT at this concentration could be used to treat the polypeptide to make the oxidized Ox-IpDf3 polypeptide unfold into a linear structure. Then, the antibacterial activity of DTT-treated Ox-IpDf3 and untreated Ox-IpDf3 against 2 strains of Staphylococcus aureus (AB94004, ATCC204036) and 2 strains of Staphylococcus epidermidis (AB208187, AB208188) was determined. S. aureus AB94004, ATCC204036) and 2 strains of Staphylococcus epidermidis (AB208187, AB208188) was determined. S. epidermidis AB94004, ATCC204036) and 2 strains of Staphylococcus epidermidis (AB208187, AB208188) was determined. Figure 5 B), compared with the Ox-IpDf3 treatment group, DTT-treated Ox-IpDf3 lost antibacterial activity at different concentrations, indicating that disulfide bond in oxidized Ox-IpDf3 polypeptide is very important for its antibacterial activity.

[0053] 3, Effect of cysteine mutation on antibacterial activity of IpDf3 Site-directed mutagenesis of cysteine residues in polypeptide molecules can directly destroy disulfide bond structure. With the aid of chemical synthesis and random oxidation method, three mutant polypeptides of IpDf3 (C4-A, C11-A, C15-A) were obtained Figure 6 A). RP-HPLC results showed that the three mutant polypeptides were single absorption peaks, indicating that the purity of the synthesized polypeptides was high. Secondly, due to the change of sequence structure, the retention time of the elution peak of the three mutants was shifted compared with IpDf3 (18.3 min), that is, the elution time of mutant IpDf3 (C4-A) was 20.4 min (Fig. 5B), the elution time of IpDf3 (C11-A) was 19.2 min (Fig. 5C), and the elution time of IpDf3 (C15-A) was 18.7 min (Fig. 5D). Figure 6 Figure 6

[0054] The results of antibacterial experiments showed that with the destruction of disulfide bonds between polypeptide molecules, the antibacterial activity of the three IpDf3 mutant polypeptides against S. aureus AB94004, S. epidermidis AB208188 and E. coli ATCC25922 was greatly reduced, and the MIC value against the two Gram-positive bacteria was also increased to 8-16 μM (Fig. 5E). These results again proved that disulfide bonds can affect the antibacterial bioactivity of IpDf3. Figure 6

[0055] Example 4, Bactericidal kinetics and bactericidal mechanism of Ipd f3, a hard tick defense peptide, against Gram-positive and negative bacteria The recovered bacterial solution was diluted 100 times with fresh MHB liquid medium to make its concentration reach about 10 7 CFU / mL. Then, the bacterial solution was treated with IpDf3 at a final concentration of 3x and 5x MIC, respectively, and positive control group (vancomycin / kanamycin) and negative control group (0.9% physiological saline) were set. Then, 200 μL of each group sample was taken at 0 min, 5 min, 15 min, 30 min, 60 min, respectively, and centrifuged at 4000 rpm for 5 min, and the supernatant was discarded, and then resuspended with 200 μL of sterile physiological saline. After 6 times of 10-fold gradient dilution, 10 μL of each group of bacterial solution was uniformly coated on LB solid medium plate, and placed in a 37 ℃ bacterial incubator for overnight culture. Finally, the plates with appropriate number of colonies (40-200) were selected for colony count, the corresponding bacterial concentration was calculated according to the dilution times, and the statistical results were analyzed by GraphPad and the curve was drawn.

[0056] ​​​The change of the membrane morphology of the bacteria after the treatment of the Ixodes persulcatus defense peptide was observed by using a scanning electron microscope (SEM), and the bactericidal mechanism of the Ixodes persulcatus defense peptide was further explored. The sample preparation steps of the scanning electron microscope are as follows: (1) sampling: 1 mL of bacterial liquid was cultured to OD630 of 0.25, 3 MIC concentration of polypeptide IpDf3 was added for incubation for 30 min, 1 h and 2 h, respectively, then centrifuged and discarded the culture medium, and the bacterial body was washed with normal saline for 3-5 times; (2) fixation: 500 μL of 2.5% glutaraldehyde fixing solution was added for resuspension, the bacterial liquid was coated on a cover glass, and the cover glass was dried at 37 ℃ for 2 h, then the cover glass was fixed with fresh 2.5% glutaraldehyde (100 mM PBS pH 7.4 dilution) overnight; (3) washing: the fixing solution was sucked off, and the cover glass was washed with PBS for 3 times, each time for 15 min; (4) gradient dehydration: 30%, 50%, 70%, 80%, 90%, and 100% ethanol gradient dehydration, each time for 15 min, and 100% ethanol was repeated once; (5) freeze-drying: the treated material was loaded into a sample basket and placed in a carbon dioxide critical point drying instrument; (6) gold spraying: the dried sample was adhered to a sample stage with conductive glue, and then was placed in an ion sputtering instrument for gold spraying; (7) scanning electron microscope observation and photographing. The results are shown in Figure 7 .

[0057] As shown in Figure 7 A, compared with the negative control group, the number of bacterial colonies of the IpDf3 treatment groups of different concentrations will obviously decrease with the extension of time, and the bactericidal speed of 5 MIC concentration IpDf3 on S. epidermidis AB208188 is faster within 15 min. There are anions in the bacterial membrane, and the amphiphilic defense peptide with positive ions is combined with the negative charge on the bacterial surface, and then inserted into the hydrophobic region of the lipid membrane to cause the destruction and decomposition of the membrane.

[0058] As shown in Figure 7 B, after the bacteria were co-cultured with the IpDf3 polypeptide for 30 min, the surface of individual bacteria appeared shrinkage and became irregular; after co-culturing for 2 h, the morphological change was serious, irregular protrusions and depressions appeared on the surface of the bacteria, and individual bacteria had the phenomenon of cell membrane rupture and content leakage, which indicated that the IpDf3 polypeptide had a direct bactericidal effect. In conclusion, the Ixodes persulcatus defense peptide IpDf3 may exert the antibacterial performance by directly destroying the membrane.

[0059] In conclusion, the present application has application value: (1) Broad coverage: efficient killing of both Gram-positive (e.g., Se 208188) and Gram-negative (e.g., Ec25922) bacteria.

[0060] (2) Clinical potential: superior to vancomycin against drug-resistant Gram-positive bacteria (MIC 0.0625 μM), and breakthrough against Gram-negative outer membrane barrier (difficult for traditional defensins).

[0061] (3) Development directions: wound spray for infections (rapid clearance of complex bacterial communities), catheter coating (prevention of biofilm formation).

[0062] Finally, it is to be understood that the term such as "including", "comprising", and "having" or variations thereof herein, are intended to be open-ended terms that specify the presence of the stated elements or features, but do not preclude the presence or addition of one or more other elements or features. Although preferred embodiments of the application have been described, a person of ordinary skill in the art will readily comprehend that the application is practiced with modifications and alterations limited only by the scope of the appended claims. Accordingly, the appended claims are intended to encompass all such modifications and alterations.

[0063] Obviously, various modifications and changes are possible in the present application without departing from the scope and spirit of the application. Accordingly, it is intended that all possible modifications and alterations be included within the scope of the present application as disclosed in the above description and the appended claims.

Claims

1. A novel defensin polypeptide of full-gorge hard tick origin, characterized in that, The novel defensin polypeptide is IpDf3, and the amino acid sequence is shown as SEQ ID NO:

1. 2.A nucleic acid molecule encoding the polypeptide of claim 1. 3.A recombinant expression vector containing the nucleic acid molecule of claim 2. 4.A recombinant bacterium or an engineered host cell line comprising the recombinant expression vector of claim 3.

5. A method for preparing the defensin polypeptide according to claim 1, characterized in that, The method comprises: cloning a gene fragment encoding the novel defensin polypeptide IpDf3 into a vector to obtain a recombinant vector; inducing expression of the recombinant vector, and obtaining the defensin polypeptide through purification. 6.The use of the polypeptide of claim 1 in the preparation of a drug for resisting gram-positive bacteria and / or gram-negative bacteria.

7. Use according to claim 6, characterized in that, The gram-positive bacteria include at least one of Staphylococcus aureus and Staphylococcus epidermidis.

8. Use according to claim 6, characterized in that, The gram-negative bacteria include at least one of Escherichia coli ATCC8739 and Escherichia coli ATCC25922.

9. An antibacterial pharmaceutical composition, characterized by, The composition comprises the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

10. The antibacterial pharmaceutical composition according to claim 9, wherein The dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections or dispersions.

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