CN111171159A

CN111171159A - Antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacteria infection as well as preparation method and application thereof

Info

Publication number: CN111171159A
Application number: CN202010069445.0A
Authority: CN
Inventors: 岳冰; 聂彬恩; 霍市城
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2020-05-19

Abstract

The invention discloses a novel antibacterial peptide for resisting infection of planococcus and intracellular bacteria and application thereof, wherein a bioactive fragment KR-12 of a human-derived antibacterial peptide LL-37 and a cell penetrating peptide TAT are combined together by a chemical covalent grafting method to form the novel antibacterial peptide TAT-KR-12. The newly synthesized antibacterial peptide TAT-KR-12 not only has the function of resisting planktonic bacteria, but also has the effect of resisting intracellular bacteria while having good biocompatibility, and in addition, has the anti-inflammatory effect. The antibacterial peptide has the advantages of simple preparation process, high efficiency, good repeatability, effective resistance to planktonic bacteria, good effect on intracellular bacteria, certain anti-inflammation, capacity of making up the defects of the traditional antibacterial drugs, and very important clinical significance.

Description

Antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacteria infection as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an antibacterial peptide for resisting infection of floating bacteria and intracellular bacteria, and a preparation method and application thereof.

Background

Staphylococcus aureus is the most common bacterium in orthopedic infections (Rumian, L., et al, Mater Sci EngCMater Biol Appl,2016.69: p.856-64.; Wright, J.A. and S.P.Nair, Int J Med Microbiol,2010.300(2-3): p.193-204.) Staphylococcus aureus invades host cells, including osteoblasts and macrophages, and is able to survive and multiply in the host cells for a period of time, thus constituting a reservoir (chwarz-Linek, U.M.) that may lead to recurrent infections.

most of the antibiotics currently used in clinical practice have limited ability to act on intracellular bacteria, making it difficult to eliminate infection unless the treatment time of the antibiotics is prolonged, the dosage is increased or the antibiotics are used in combination, which explains why antibacterial efficacy gradually decreases and how acute infection is converted into chronic infection when β -lactam antibiotics are used to treat staphylococcus aureus infection, although macrolide antibiotics are able to penetrate cells, the efficiency of conventional antibiotics to treat staphylococcus aureus infection is decreasing (mr, n., et al, antibiotic, agentschemicother, 1994.38(12): p.2738-42.), and thus a new antibacterial agent is required which, in addition to anti-planktonic bacteria, should also be effective against intracellular bacteria while not damaging host cells.

Antimicrobial peptides are effective at low concentrations against a variety of microorganisms, including microorganisms that are resistant in many cases to traditional antibiotics. Traditional antibiotics kill or inhibit bacterial growth by targeting various biosynthetic processes in bacterial growth (including protein, RNA, DNA, peptidoglycan, and folate synthesis, etc.), while antimicrobial peptides are generally not directed to specific microbial molecules, but interact directly with microbial membranes and permeate rapidly, in other words, they act primarily through lytic mechanisms. Their amino acid composition determines their structural properties, such as amphiphilicity, cationic charge, shape and size, facilitating interaction with the microbial surface, insertion of lipid bilayers, and thus destabilization and compromising the physical integrity of the bacterial membrane. Several different models have been proposed to describe the consequences of this mode of action, including the mechanism of barrel-shaped walls and annular apertures, formation of channels of aggregation and detergent-like carpet effects (Brogden, K.A., Nat Rev Microbiol,2005.3(3): p.238-50.).

Compared with the traditional antibiotics, the antibacterial peptide has broad-spectrum antibacterial performance and low toxicity, and also has other biological activities such as regulating the immunity of an organism and the like. The antibacterial peptide can be directly antibacterial and can eliminate bacteria by regulating a defense system of an organism, the bacteria are not easy to generate drug resistance, the antibacterial peptide can enter cells to play an immunoregulation role, and the antibacterial peptide has complex and unknown biological activity.

Disclosure of Invention

The invention aims to provide an antibacterial peptide for resisting planktonic bacteria and intracellular bacterial infection, a preparation method and application thereof, and solves the problem that the traditional antibacterial drugs cannot effectively resist intracellular bacteria.

In order to achieve the aim, the invention provides an antibacterial peptide TAT-KR-12 for resisting the infection of the phytoplankton and the intracellular bacteria, which is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.

The invention also provides a preparation method of the antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacterial infection, which is characterized in that the antibacterial peptide TAT-KR-12 is obtained by joining a bioactive fragment KR-12 of the human-derived antibacterial peptide LL-37 and a cell penetrating peptide TAT together through chemical covalent grafting.

Preferably, the sequence of the biologically active fragment KR-12 is as shown in SEQ ID NO: 2, respectively.

Preferably, the sequence of the cell penetrating peptide TAT is as set forth in SEQ ID NO: 3, the sequence is loaded with a positive charge, and its membrane-penetrating ability is independent of classical endocytosis.

Preferably, the sequence of the human-derived antibacterial peptide LL-37 is shown as SEQ ID NO: 4, respectively.

Preferably, said chemical covalent grafting is in particular: the biologically active fragment KR-12 of the human-derived antibacterial peptide LL-37 and the cell-penetrating peptide TAT were prepared by solid phase polypeptide synthesis, followed by purification of the polypeptide by High Performance Liquid Chromatography (HPLC).

The invention also provides application of the antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacterial infection in preparing anti-planktonic bacteria medicaments.

The invention also provides application of the antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacterial infection in preparing a medicament for resisting intracellular bacterial infection.

Preferably, the type of intracellular bacteria used in the application is staphylococcus aureus (ATCC 25923), which is capable of entering the cell and surviving inside the cell for a period of time while having the ability to infect.

The invention also provides the function of the antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacteria infection in preparing a medicament for inhibiting inflammatory reaction.

preferably, the inhibition of the inflammatory response is inhibition of macrophage inflammatory response, in particular inhibition of the expression of inflammatory factors tumor necrosis factor (TNF- α), interleukin-1 β (IL-1 β), Inducible Nitric Oxide Synthase (INOS) and related inflammatory genes.

More preferably, the macrophage is mouse monocyte macrophage leukemia cell RAW264.7, which is derived from a tumor induced by a leukemia virus of the murine family Male Abelson.

The sequence of the antibacterial peptide KR-12 is KRIVQRIKDFLR, which is a bioactive fragment derived from the human antibacterial peptide LL-37. The antimicrobial peptide KR-12 has broad-spectrum antimicrobial capability and no cytotoxicity to mesenchymal stem cells. The cell penetrating peptide TAT (sequence YGRKKRRQRRR, HIV-1TAT protein residues 47-57) is loaded with positive charges, and the membrane penetrating capability of the cell penetrating peptide TAT is independent of classical endocytosis. Has been widely used for intracellular delivery of membrane impermeable biomacromolecules.

The internalization of bacteria into cells is the main reason of chronic infection and reinfection, most of antibacterial drugs are ineffective to the bacteria in the cells at present, and the invention has the effect of resisting the intracellular bacteria while not damaging the cells by using the novel antibacterial peptide TAT-KR-12 prepared by a solid-phase polypeptide synthesis method through the antibacterial peptide KR-12 and the penetration TAT.

The invention has the beneficial effects that:

(1) the invention provides a novel antibacterial peptide for resisting infection of planococcus and intracellular bacteria and application thereof, wherein a bioactive fragment KR-12 of a human-derived antibacterial peptide LL-37 and a cell penetrating peptide TAT are combined together by a chemical covalent grafting method to form the novel antibacterial peptide TAT-KR-12. The newly synthesized antibacterial peptide TAT-KR-12 not only has the function of resisting planktonic bacteria, but also has the effect of resisting intracellular bacteria while having good biocompatibility, and in addition, the novel antibacterial peptide also has the anti-inflammatory effect.

(3) The preparation method of the novel antibacterial peptide is a solid-phase polypeptide synthesis method, wherein an insoluble high polymer is used as a carrier, one reactant is fixed on the high polymer carrier through an active group of the insoluble high polymer, so that organic synthesis is carried out on a solid phase, and the polypeptide is purified by High Performance Liquid Chromatography (HPLC) after synthesis, wherein the purity is over 95 percent. The method has the advantages of mature technology, simple process, high efficiency and low cost, and can synthesize the polypeptide with any monomer sequence.

(3) The antibacterial peptide product of the invention does not produce hemolytic property, has good biocompatibility and wide antibacterial spectrum, and has better antibacterial effect on gram-positive bacteria, gram-negative bacteria and drug-resistant strains. More importantly, aiming at the defect that clinically common antibacterial drugs can not effectively resist intracellular bacteria, the novel antibacterial peptide has good effect of resisting the intracellular bacteria and also has anti-inflammatory property. Therefore, the antibacterial peptide of the product can be better applied to the treatment of clinical intracellular bacterial infection.

Drawings

FIG. 1: helical projection of the antimicrobial peptides KR-12, TAT and TAT-KR-12 of the present invention;

FIG. 2: the hemolytic activity detection results of the antimicrobial peptides KR-12, TAT and TAT-KR-12 of the invention;

FIG. 3: the cytotoxicity detection results of the antimicrobial peptide KR-12, TAT and TAT-KR-12 are shown in the specification;

FIG. 4: cell plasma membrane depolarization results of the antimicrobial peptides KR-12, TAT and TAT-KR-12 of the present invention;

FIG. 5: scanning electron microscope images of bacteria treated by the antibacterial peptide TAT-KR-12; wherein, A is before the treatment of staphylococcus aureus, B is after the treatment of staphylococcus aureus by TAT-KR-12(2 × MIC) for 2 hours, C is before the treatment of escherichia coli, and D is after the treatment of escherichia coli by TAT-KR-12(2 × MIC) for 2 hours;

FIG. 6: determining the result of the in vitro intracellular bacterium resistance of the antibacterial peptide TAT-KR-12; wherein, A is a statistical chart of in vitro intracellular bacteria resistance of a control group, a vancomycin group and a TAT-KR-12 group, and B is a photo of a single-cell colony growing on a bacterial culture plate;

FIG. 7 shows anti-inflammatory effects of TAT-KR-12 by Lipopolysaccharide (LPS), wherein A is a statistical graph of ELISA-measured secretion levels of inflammatory factor IL-1 β, and B is a statistical graph of ELISA-measured secretion levels of inflammatory factor TNF- α;

FIG. 8: the in vivo anti-planktonic bacteria and intracellular bacteria of the antimicrobial peptide TAT-KR-12 are determined; a is plate coating quantification of planktonic bacteria infected skin bacteria; b is a gross photograph of the mice on

days

3, 5 and 7 of the treatment cycle; c is the quantity of intracellular bacteria infected skin bacteria; d is a gross photograph of mice on

days

3, 5 and 7 of the treatment cycle.

Detailed Description

The present invention is further described below in conjunction with the following detailed description and the accompanying drawings, it being understood that the following detailed description and/or the drawings are only illustrative of the invention and are not limiting.

1. Preparation of peptide:

peptides were prepared by standard Fmoc-based solid phase synthesis techniques on Rink amide MBHA resin. Dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were used as coupling reagents and a 10-fold excess of Fmoc amino acid was added in each coupling cycle. After cleavage with a mixture of TFA/H2O/thioanisole/phenol/ethanedithiol/triisopropylsilane (81.5: 5: 5: 5: 2.5: 1, v/v/v/v/v/v/v/v) and deprotection for 2 hours at room temperature, the crude peptide was extracted repeatedly with diethyl ether and purified on an analytical Vydac C18 column (length: 250mm, internal diameter: 20mm, pore diameter:

particle size: 15mm) by reverse phaseLiquid chromatography (RP-HPLC) purification. Elution was performed using an appropriate 0-90% water/acetonitrile gradient.

The antibacterial peptide KR-12, the cell penetrating peptide TAT and the novel antibacterial peptide TAT-KR-12 are synthesized by the method, and the amino acid sequences of the peptides are as follows: KRIVQRIKDFLR (SEQ ID NO: 2), YGRKKRRQRRR (SEQ ID NO: 3), YGRKKRRQRRRKRIVQRIKDFLR (SEQ ID NO: 1). The purity of each peptide was over 95% by HPLC analysis.

FIG. 1 is a helical projection of the antimicrobial peptides KR-12, TAT and TAT-KR-12: the amino acid composition and arrangement mode of each peptide segment on a two-dimensional plane.

Table 1 shows the physicochemical properties of KR-12, TAT and TAT-KR-12.

TABLE 1

2. Determination of the antimicrobial peptides KR-12, TAT and TAT-KR-12 Minimum Inhibitory Concentration (MIC):

respectively culturing staphylococcus aureus (ATCC 25923), methicillin-resistant staphylococcus aureus (MRSA, ATCC43300) and staphylococcus epidermidis (ATCC,35984), methicillin-resistant staphylococcus epidermidis (MRSE, ATCC,287) and escherichia coli (ATCC,25922) in MHB culture medium to logarithmic growth phase, and determining the mic value of the polypeptide by adopting a modified clinical laboratory and a broth microdilution method of a standard institute to determine the in vitro antibacterial activity of the polypeptide. Adjusting the bacterial concentration to 1 × 10 by McLeod⁶CFU/mL. Immediately after the bacteria were mixed well with the solution, 100. mu.L of each bacterial suspension was taken out and placed in a 96-well plate, and the polypeptide was diluted by dissolving in 0.01% acetic acid and 0.2% Bovine Serum Albumin (BSA). mu.L of each of the antimicrobial peptides was diluted with 100. mu.L of MHB to a concentration of 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2. mu.g/mL in a 96-well plate, and 3 wells were repeated for each set, with the broth containing microbial cells as a negative control and the non-inoculated broth as a positive control. And (3) statically culturing for 24h at 37 ℃ in a constant-temperature incubator, and recording the Minimum Inhibitory Concentration (MIC) of each group of bacteria. And (4) judging a result: the wells in which no bacterial growth was detected were taken as the minimum inhibitory concentration.

Table 2 shows the results of the measurement of the minimum antimicrobial concentration and the minimum antimicrobial concentration of the peptide, and it can be seen from the table that TAT-KR-12 has significantly enhanced in vitro antimicrobial and bactericidal activity after the grafted cells penetrate the peptide. The novel antibacterial peptide TAT-KR-12 has wide antibacterial spectrum and good antibacterial effect.

TABLE 2

3. Detection of hemolytic activity of the antimicrobial peptides KR-12, TAT and TAT-KR-12:

freshly collected human blood was centrifuged with heparin to remove the buffy coat and the red blood cells obtained were washed three times with Phosphate Buffered Saline (PBS), centrifuged at 1,000g for 10 minutes and resuspended to 4% (v/v) in PBS. Fresh human red blood cells (hRBC) were washed three times with PBS (35mM phosphate buffer, 0.15M NaCl, pH7.4) and centrifuged at 1000g for 5 minutes. Two-fold serial dilutions of antimicrobial peptide in PBS (concentration tested in the range of 8, 16, 32, 64,128,256,512,1024 μ g/mL) were added to each well of a 96-well plate (total volume: 100 μ L). An equal volume (100 μ L) of 4% w/v hRBC in PBS suspension was added to each well to reach a final volume of 200 μ L. The plates were incubated at 37 ℃ for 1 hour, and then the cells were pelleted by centrifugation at 1000g for 5 minutes. The supernatant (100. mu.L) was transferred to a clear 96-well plate. Hemoglobin was detected by measuring the absorbance at 414nm (Molecular Devices, Sunnyvale, Calif., USA). The value of zero hemolysis (negative control) was determined using PBS (APBS), while 100% hemolysis (positive control) was determined using 0.1% (v/v) Triton X-100 (ATriton). Percent hemolysis was calculated as follows: homolysis% (Asample-APBS)/(A Triton X-100-APBS) × 100

FIG. 2 shows the results of measurement of hemolytic activity of peptides. The results in the figure show that TAT-KR-12 does not show any significant hemolytic activity even at concentrations as high as 1024. mu.g/mL, indicating that the novel antimicrobial peptide of the present invention has no hemolytic activity and good biocompatibility.

4. Cytotoxicity test of antimicrobial peptides KR-12, TAT and TAT-KR-12:

taking out the jelly containing RBMSCs from the liquid nitrogen tankStoring the tube, directly immersing the tube in 37 ℃ constant temperature water for incubation, shaking the tube to melt the tube as soon as possible without any time, taking the tube out after the tube is melted, spraying 70% alcohol for disinfection, putting the tube into a super clean bench, opening a cover, sucking out cell suspension by using a pipette, adding the cell suspension into a 50mL centrifuge tube, adding 10mL culture solution, uniformly mixing, centrifuging the mixture, and centrifuging the mixture at 1000rpm for 5 min. Discarding the supernatant, adding 10mL of culture solution to resuspend the cells, inoculating to a culture dish, performing static culture in an incubator at 37 ℃, replacing the culture solution once the next day, and continuing to culture. When the cells reached 80% confluence, they were collected by trypsinization and used in the experiment. CCK-8 is used for detecting the influence of the antibacterial peptide on the proliferation of RBMSCs, 100 mu L of the RBMSCs for more than 3 passages are added into each hole of a 96-hole plate, and the final cell amount is 5 multiplied by 10³One/well, putting 96-well plate into constant temperature incubator at 37 deg.C and 5% CO₂After incubation for 24h under conditions, the supernatant was carefully aspirated, 100. mu.L of antimicrobial peptide (1000, 500, 250, 100, 50, 10, 5. mu.g/mL) was added at various concentrations to each well, and fresh medium was used as a negative control. At preset time points (1d, 3d, 5d), the wells were aspirated, 100. mu.L of CCK-8 working solution was added to each well, the 96-well plate was returned to the incubator and incubated for 2h, and OD was measured at 450 nm.

FIG. 3 shows the results of the peptide cytotoxicity assay. As can be seen from the results in the figure, TAT-KR-12 concentrations up to 500. mu.g/mL did not show any significant growth inhibition at the third day as compared with the control group without the peptide. At day 5, TAT-KR-12 had good biocompatibility at a concentration of not more than 100. mu.g/mL.

5. Cytoplasmic membrane depolarization experiments:

peptide-induced depolarization activity of the bacterial plasma membrane was detected by the fluorescent dye diSC3-5(Sigma Aldrich) using a membrane potential sensitive probe. Coli ATCC 25922 bacteria were cultured in bacterial medium MHB at 37 ℃ to mid-log phase under constant shaking at 220rpm, harvested by centrifugation at 1000g for 10 minutes, and washed with washing buffer (10mM HEPES, 5mM glucose and 50. mu.g/mL)^-1In (C) is₂pH7.4) and resuspended to 2X 10 in the same buffer⁸CFU/mL. DiSC3(5) was added to a final concentration of 1.5. mu.M and incubated at ambient temperature, which was kept protected from light, to allow dye uptake andthe fluorescence is quenched. After 30 minutes, bacterial cells were diluted 50-fold in assay buffer, then 45 μ Ι _ was added to 384-well black polystyrene plates (Corning, CLS 3573). TAT-KR-12(0.25, 1, 4 XMIC) was added to the bacterial suspension and used

The m1000 Pro Multi-mode microplate reader monitors the fluorescence change for 30 minutes. Membrane depolarization was monitored by the change in fluorescence intensity emitted by dicc 3-5 (excitation λ 622nm, emission λ 670nm) after addition of different concentrations of peptide. The ability of the peptides to depolarize the membrane was measured in the presence of the membrane-selective fluorescent probe dicc 3 (5). Dicc 3(5), a fluorescent probe, was focused on the energized membrane and its fluorescence was affected by the gradient of the membrane potential, thereby increasing its emission intensity.

FIG. 4 shows the result of depolarization of the plasma membrane. As can be seen from the results in the figure, no increase in fluorescence was observed after KR-12 treatment; in contrast, there was a clear change in fluorescence intensity over time after treatment with TAT-KR-12, indicating that TAT-KR-12 was able to depolarize the plasma membrane of the cell.

6. Detecting the damage of the antibacterial peptide TAT-KR-12 to the bacterial cell membrane by a scanning electron microscope:

for scanning electron microscope sample preparation, the bacteria Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC25923 were cultured in bacterial medium MHB under constant shaking at 220rpm at 37 ℃ to mid-log phase, harvested by centrifugation at 1000g for 10 minutes, washed three times and diluted with 10mM PBS to an OD600 value of 0.2. Cells were incubated at 37 ℃ for 120 min with the experimental peptide concentration 2 × MIC and the control group added with an equal amount of PBS. Then, the cells were harvested by centrifugation at 5000g for 5 minutes, washed three times with PBS, fixed with 2.5% (w/v) glutaraldehyde at 4 ℃ overnight, and then washed twice with PBS (pH 7.2). Cells were dehydrated with gradient ethanol (50%, 70%, 80%, 90% and 100%) for 10 min and in 100% ethanol for 15 min. Then, the cells were transferred in 100% ethanol and a mixture of t-butanol and dry t-butanol (v: v ═ 1: 1) for 15 minutes. Finally, the samples were dried in a critical point desiccator with liquid CO₂Dehydrating, coating with gold palladium, and scanningElectron microscopy (Hitachi S-4800, SEM).

FIG. 5 is a scanning electron micrograph of bacteria treated with TAT-KR-12. After 2 hours of TAT-KR-12(2 XMIC), some of the cell membranes of Staphylococcus aureus and Escherichia coli were completely destroyed and morphology was completely lost, while the untreated group showed an intact cell membrane structure.

7. In vitro intracellular bacterium resisting effect determination of antibacterial peptide TAT-KR-12:

the cells used in this section were RAW264.7 macrophages. About 5X 10⁵RAW264.7 in 24-well plates, cells were seeded at 37 ℃ in a medium containing 5% CO₂Was cultured overnight in a cell culture chamber to ensure cell adhesion. Staphylococcus aureus ATCC25923 bacteria were cultured in MHB at 220rpm at 37 ℃ under constant shaking to the mid-log phase, harvested by centrifugation at 1000g for 10 minutes, washed three times, and quantified to 3X 10⁸CFU/mL. About 13.3 μ L (multiplicity of infection 10) of the quantified bacteria infected RAW264.7 cells for 1 hour. Subsequently, the cells were washed gently with ice PBS for 2 times, and then cultured in a medium containing 50. mu.g/mL gentamicin for 2 hours. After removing gentamicin, the PBS group was used as a negative control group, the vancomycin (10MIC) group was used as a positive control group, and the antimicrobial peptide TAT-KR-12(10MIC) group was used as an experimental group, and the incubation was performed for 6 hours and 24 hours again. At the pre-set time point, cells were lysed on Triton X-100 ice for 15 minutes. And (3) performing gradient dilution to a proper multiple by using ice PBS, coating the lysate on a bacterial culture plate, culturing for 24 hours at 37 ℃, counting the number of single-cell colonies growing on the bacterial culture plate, and comparing the intracellular bacterial resistance effects of the antibacterial peptides TAT-KR-12 and vancomycin at different time points.

FIG. 6 shows the results of TAT-KR-12 determination against intracellular bacteria in vitro. As can be seen from the figure, after 6h and 24h of treatment, the vancomycin group has poor intracellular bacterium resistance effect and has no statistical difference with the PBS treatment group, and TAT-KR-12 can obviously inhibit the propagation of intracellular bacteria and has good intracellular bacterium resistance effect. The novel antibacterial peptide of the invention can effectively resist intracellular bacteria in vitro.

Effect of TAT-KR-12 on Lipopolysaccharide (LPS) -induced cytokines:

RAW264.7 cells were plated at 5X 10⁵after that, the cell was added with the antimicrobial peptide TAT-KR-12, the control group was added with sterile PBS, followed by incubation for 1 hour, and then stimulated with LPS (100ng/mL) at 37 ℃ for 15-18 h.the cell supernatant was collected at a predetermined time, and the secretion levels of inflammatory cytokines TNF- α and IL-1 β in the sample were measured by ELISA, according to the manufacturer's instructions, the detection limit of the measurement was 0.12-1000 pg/mL for TNF- α and 1.5-1000 pg/mL for IL-1 β in the case of the assay, the concentration of the inhibitor was determined by the concentration of the inhibitor in the culture medium.

FIG. 7 is an anti-inflammatory effect of TAT-KR-12 by Lipopolysaccharide (LPS) secretion levels of TNF- α and IL-1 β, and from the results, it can be seen that TAT-KR-12 is effective in inhibiting secretion of TNF- α and IL-1 β as compared to LPS group without peptide, indicating that TAT-KR-12 has anti-inflammatory properties in vitro.

TAT-KR-12 in vivo anti-planktonic and intracellular bacteria assay:

6-8 week old female BALB/c mice (25-30 g in weight) were purchased from Shanghai Slacca for all experiments.

(1) Model of planktonic bacteria subcutaneous infection

For the skin on the back of the mice, the mice were anesthetized by intraperitoneal injection of 3% pentobarbital (1mL/kg-1) prior to surgery and placed on sterile drape to provide sterile conditions during surgery. Thereafter, the mice were shaved on the back and wiped with 70% chlorhexidine alcohol and randomly assigned to the appropriate study group (5 mice per group). Next, the subcutaneous injection is autoclaved with equal volume

1 microcarrier microspheres (131 to 220 um; Sigma-Aldrich, USA) mixed 5X 10⁷CFU/flanking strain. One hour after the inoculation of the bacteria, TAT-KR-12(20mg/kg) was injected subcutaneously near the abscess to determine its anti-planococcal effect in vivo, and sterile physiological saline was used as a negative control; vancomycin (20mg/kg) was used as a positive control.

(2) Intracellular bacterial subcutaneous infection model

For intracellular bacterial infection studies, 100. mu.L of 5X 10 was injected intraperitoneally⁸CFU/mL S.aureus infection is smallMice. The abdominal infected mice were sacrificed 24 hours later and the peritoneum was rinsed with 5mL of pre-cooled PBS. The peritoneal washing solution was centrifuged for 5 minutes in a bench top centrifuge at 1,500 r.p.m., 4 ℃. Peritoneal macrophages were harvested and the cells were treated with 50 μ g/mL lysostaphin for 20 minutes at 37 ℃ to kill contaminating extracellular bacteria. Peritoneal cells from donor mice were washed 3 times with pre-cooled PBS to remove lysostaphin and the cells were pooled by centrifugation; cells from five donors were injected subcutaneously into the back of recipient mice. One hour after cell inoculation, TAT-KR-12(20mg/kg) was injected subcutaneously near the abscess to determine its anti-planococcal effect in vivo, and sterile physiological saline was used as a negative control; vancomycin (20mg/kg) was used as a positive control.

The medicine is administered once every 24 hours for seven days. To evaluate the therapeutic effect of TAT-KR-12 on planktonic and intracellular bacterial infections in mice, the body weight of the mice was examined daily and wounds were observed regularly and photographed. Mice were sacrificed at the end of treatment. To determine the bacterial count in infected tissue samples, the dorsal abscess skin was aseptically excised and homogenized in physiological saline (1.0mL), then plated on TSA agar plates, incubated at 37 ℃ for 24 hours, and the bacterial count at the abscess sites was counted.

(3) Determination of the Effect of TAT-KR-12 on in vivo resistance to Aeromonas

FIG. 8 is a determination of TAT-KR-12 against planktonic and intracellular bacteria in vivo. As shown in FIG. 8, A is the plankton-infected skin bacteria plating quantification, and the bacteria at the infected part of TAT-KR-12 treated group are obviously reduced compared with the untreated control group; b is a gross photograph of mice on

days

3, 5 and 7 during the treatment cycle: mice treated with vancomycin and TAT-KR-12 had smaller necrotic areas of skin lesions and healed more rapidly, whereas the affected areas in the control group had severely purulent, ulcerated skin.

(4) Determination of the in vivo Effect of TAT-KR-12 against intracellular bacteria

As shown in FIG. 8, C is the quantification of intracellular bacteria infected skin bacteria plated, and TAT-KR-12 treated group showed a significant reduction in bacteria at the infected site compared to untreated control group. D is a gross photograph of mice on

days

3, 5 and 7 during the treatment cycle: the mice in TAT-KR-12 treated group had smaller necrotic area of skin lesion and faster healing of lesion, while the skin of the affected part in the untreated control group was severely suppurative, and the infection was inhibited but less effective than in TAT-KR-12 treated group by vancomycin treatment at a high concentration, and abscess remained in the gross photograph at 7 days.

SEQUENCE LISTING

<110> Shanghai university of traffic medical college affiliated renji hospital

<120> antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacterial infection, and preparation method and application thereof

<130>PCN1193470

<160>4

<170>PatentIn version 3.5

<210>1

<211>23

<212>PRT

<213>Artificial Sequence

<220>

<223>TAT-KR-12

<400>1

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Arg Ile Val Gln

1 5 10 15

Arg Ile Lys Asp Phe Leu Arg

20

<210>2

<211>12

<212>PRT

<213>Homo sapiens

<400>2

Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg

1 5 10

<210>3

<211>12

<212>PRT

<213>Human immunodeficiency virus type 1

<400>3

Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg

1 5 10

<210>4

<211>37

<212>PRT

<213>Homo sapiens

<400>4

Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu

1 5 10 15

Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val

20 25 30

Pro Arg Thr Glu Ser

35

Claims

1. An antibacterial peptide TAT-KR-12 for resisting infection of phytoplankton and intracellular bacteria, wherein the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. The method for preparing the antibacterial peptide TAT-KR-12 resisting the infection of the phytoplankton and the intracellular bacteria as claimed in claim 1, wherein the antibacterial peptide TAT-KR-12 is obtained by joining the bioactive fragment KR-12 of the human-derived antibacterial peptide LL-37 and the cell penetrating peptide TAT together through chemical covalent grafting.

3. The method for preparing the antibacterial peptide TAT-KR-12 resisting the infection of the phytoplankton and the intracellular bacteria as claimed in claim 2, wherein the sequence of the bioactive fragment KR-12 is shown as SEQ ID NO: 2, respectively.

4. The method of claim 2, wherein the sequence of the cell penetrating peptide TAT is as shown in SEQ ID NO: 3, respectively.

5. The method for preparing the antibacterial peptide TAT-KR-12 for resisting the infection of the phytoplankton and the intracellular bacteria as claimed in claim 2, wherein the sequence of the human-derived antibacterial peptide LL-37 is shown as SEQ ID NO: 4, respectively.

6. The method for preparing the antibacterial peptide TAT-KR-12 for resisting the infection of the phytoplankton and the intracellular bacteria according to claim 2, wherein the chemical covalent grafting is specifically as follows: the biologically active fragment KR-12 of the human-derived antibacterial peptide LL-37 and the cell-penetrating peptide TAT were prepared by solid phase peptide synthesis, followed by purification of the polypeptide by high performance liquid chromatography.

7. Use of the antibacterial peptide TAT-KR-12 against infections by phytoplankton and intracellular bacteria according to claim 1 for the preparation of an anti-phytoplankton medicament.

8. Use of the antibacterial peptide TAT-KR-12 against infections by phytoplankton and intracellular bacteria according to claim 1 in the preparation of a medicament against infections by intracellular bacteria.

9. The use of the anti-bacterial peptide TAT-KR-12 against infections by phytoplankton and intracellular bacteria according to claim 8, in the manufacture of a medicament against infections by intracellular bacteria, wherein the type of intracellular bacteria in said use is staphylococcus aureus.

10. Use of the antibacterial peptide TAT-KR-12 against infections by phytoplankton and intracellular bacteria according to claim 1 for the preparation of a medicament for inhibiting inflammatory reactions.

11. the use of the antibacterial peptide TAT-KR-12 against infections by phytoplankton and intracellular bacteria according to claim 10, in the manufacture of a medicament for inhibiting inflammatory responses, wherein said inhibition of inflammatory responses is inhibition of macrophage inflammatory responses, in particular inhibition of secretion of inflammatory factors TNF- α, IL-1 β and related inflammatory factors.

12. The use of the anti-bacterial peptide TAT-KR-12 against infections by phytoplankton and intracellular bacteria for the manufacture of a medicament for inhibiting inflammatory response according to claim 11, wherein said macrophage is mouse mononuclear macrophage leukemia cell RAW264.7, which is derived from a tumor induced by the leukemia virus of the murine family male Abelson.