Ts-ELP temperature-sensitive self-assembly nano fusion protein, antibiotic delivery system and preparation method

Abstract

The invention discloses a Ts-ELP temperature-sensitive self-assembly nano fusion protein, an antibiotic delivery system and a preparation method, and belongs to the technical field of nano preparations. The fusion protein comprises antibacterial peptide Ts and elastin-like polypeptide ELP which are sequentially connected. An MMP-9 restriction enzyme cutting site is introduced between the antibacterial peptide Ts and the elastin-like polypeptide ELP. Fusion protein and tigecycline (Tig) are self-assembled to form an antibiotic delivery system, the delivery system has Gram-negative bacterium surface lipopolysaccharide targeted recognition capability, and drug release is triggered through pH response and a phase separation mechanism in an infection microenvironment, so that the enrichment efficiency of tigecycline in a focus area is enhanced, and the drug delivery efficiency is improved. The antibiotic delivery system has the technical advantages of strong targeting property, enhanced antibacterial activity, low drug-resistant induction risk, good biological safety, simple preparation process, high yield and the like, can be used for treatment of multi-drug-resistant bacterium infection, and is especially suitable for prevention and treatment of carbapenem-resistant klebsiella pneumoniae (CRKP) and drug-resistant gram-negative bacterium infection related diseases.

Description

Technical Field

[0001] This invention belongs to the field of nano-formulation technology, specifically relating to Ts-ELP thermosensitive self-assembled nanofusion protein, antibiotic delivery system and preparation method. Background Technology

[0002] In recent years, infections caused by multidrug-resistant Gram-negative bacteria, especially carbapenem-resistant Klebsiella pneumoniae (CRKP), have become a serious public health problem. While tigecycline, currently used to treat CRKP, has a broad antibacterial spectrum, it suffers from drawbacks such as weak tissue penetration, a short half-life in vivo, and the potential for toxic side effects at high concentrations. Furthermore, its free form is difficult to accumulate at the site of infection, leading to unstable efficacy.

[0003] In recent years, nanomedicine delivery systems based on protein engineering and self-assembly mechanisms have gradually shown advantages, but existing delivery systems generally suffer from the following problems: (1) lack of Gram-negative bacteria structure-specific recognition ability; (2) unstable drug loading process or difficulty in large-scale expression; and (3) lack of infection microenvironment response and controlled release ability, resulting in drug release in healthy tissues as well.

[0004] Therefore, there is an urgent need to develop a delivery system with pathogen targeting, structural stability, thermoresponsiveness, and controllable drug release capabilities to improve the efficacy of tigecycline and reduce the risk of drug resistance and toxicity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide Ts-ELP temperature-sensitive self-assembled nanofusion protein, antibiotic delivery system, and preparation method, thereby solving the problems in the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions: Ts-ELP thermosensitive self-assembled nanofusion protein includes: an antimicrobial peptide Ts and an elastin-like polypeptide ELP connected in sequence; an MMP-9 restriction site is introduced between the antimicrobial peptide Ts and the elastin-like polypeptide ELP.

[0007] Furthermore, the sequence of the elastin-like polypeptide ELP is: (VPG(VH4)G)n, where n is a natural number from 3 to 100.

[0008] Furthermore, the MMP-9 restriction site is any amino acid sequence including GPLGLPG.

[0009] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0010] The above-mentioned method for preparing Ts-ELP thermo-sensitive self-assembled nanofusion protein includes the following steps: Antimicrobial peptide Ts and elastin-like polypeptide ELP were genetically recombined using DNA recombination technology to construct recombinant plasmids. The constructed recombinant plasmid was transformed into competent BL21(DE3) cells, and the expression of the fusion protein was induced by isopropyl-β-D-thiogalactoside. The bacterial cells were lysed and the proteins were purified. The purified protein was digested using thrombin. The enzyme-digested protein was then purified a second time to obtain the target fusion protein.

[0011] The above-mentioned fusion protein is used in the preparation of antibacterial drugs or drug carriers for Gram-negative bacillus infections.

[0012] Furthermore, the Gram-negative bacillus is either CRKP or KPN.

[0013] A drug carrier comprising the aforementioned fusion protein.

[0014] A nano-antibiotic delivery system includes the aforementioned fusion protein and tigecycline loaded onto the fusion protein.

[0015] The preparation method of the above-mentioned nano-antibiotic delivery system includes: incubating the fusion protein with tigecycline at 50°C, and then adding ZnCl2 to self-assemble and form a nano-antibiotic delivery system.

[0016] The beneficial effects of this invention are: 1. The delivery system of the present invention integrates a targeted antimicrobial peptide Ts, which specifically binds to bacterial surface lipopolysaccharides and can specifically recognize and bind to the surface of Gram-negative bacteria; it also integrates a human-derived polypeptide ELP, which has good biocompatibility. As a drug delivery system, it has the general characteristics of macromolecular polymer drug delivery systems, such as passively targeting the infection site through the EPR effect, and actively targeting the carrier by adding a targeting ligand, effectively prolonging the drug half-life, reducing drug toxicity, increasing drug solubility, and other advantages. This allows the delivery system to effectively avoid the immune system reducing the recognition and clearance of the reticuloendothelial system (RES) and increasing the drug half-life. 2. The present invention has the characteristics of acid-sensitive release and enzyme responsiveness, and can rapidly release tigecycline and antimicrobial peptide Ts in the low pH environment and in the presence of high concentration of MMP-9 enzyme at the bacterial infection site.

[0017] 3. The nano-antibiotic of the present invention has a nanoscale size, regular morphology, and high yield and purity; after intravenous injection, it delivers tigecycline and antimicrobial peptides to the bacterial infection site through blood circulation, thereby playing a dual bactericidal role and reducing the risk of drug resistance induction. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the method for constructing the antibiotic delivery system of the present invention; Figure 2 This is an SDS-PAGE gel electrophoresis image of the purified Ts-ELP fusion protein. Figure 3 The graph shows the multi-peak distribution of Tig@TsE particles at different pH values. Figure 4 Transmission electron microscopy images of Ts-ELP and Tig@TsE; Figure 5 The results of drug release experiments of Tig@TsE at different pH values; Figure 6 The results are from the Tig@TsE in vitro bacterial binding assay. Figure 7 The results are from the in vitro antibacterial test of Tig@TsE. Figure 8 The experimental results show that Tig@TsE reduces the induction of drug resistance; Figure 9 The in vivo targeting effect of Tig@TsE; Figure 10 Image showing the therapeutic effect of tail vein administration in a BALB / c mouse model of acute pneumonia. Figure 11 This is a schematic diagram illustrating the preparation and antibacterial mechanism of nano-antibiotics. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 This embodiment describes the design and construction of the Ts-ELP fusion protein sequence; The prokaryotic expression plasmid pET-32a(+)-Thrombin-Ts-Linker-ELP encoding Ts-ELP was synthesized using a whole-genome synthesis method. The complete amino acid sequence is as follows: A thrombin restriction site (LVPRGS) and the amino acid sequence of the natural antimicrobial peptide Ts, as shown in SEQ ID NO.2 (specifically GSKKPVPIIYCNRRTGKCQRM), were introduced downstream of the TrxA tag in the pET-32a(+) vector. The Ts sequence was fused to the C' terminus of the MMP-9 restriction site (GPLGLPG) and the N' terminus of pH-sensitive human elastin peptides (ELPs) via a linker (GGGSG). A His tag was introduced at the C' terminus of the ELP as a purification tag for the fusion protein. The complete sequence of the Ts-ELP fusion protein is shown in SEQ ID NO.1. The amino acid sequence was optimized using E. coli codons. Thrombin-Ts-Linker-ELP, synthesized conventionally, was cloned into the pET-32a(+) (Ampicillin) vector by adding 5' MScI and 3' XhoI DNA restriction sites, preparing recombinant plasmid DNA (pET-32a(+)-Thrombin-Ts-Linker-ELP) and BL21(DE3) stab strain containing this recombinant plasmid. Two cysteine ​​residues (Cys, C) in the Ts sequence were mutated to alanine residues (Ala, A), and the mutant non-targeting vector pET-32a-Thrombin-MTs-Linker-ELP (MTs-ELP) expression plasmid and the corresponding stab strain served as a non-targeting control group. A schematic diagram of the fusion protein construction method is shown below. Figure 1 .

[0022] In this embodiment, the complete sequence of the Ts-ELP fusion protein (SEQ ID NO.1) is as follows: GSKKPVPIIYCNRRTGKCQRMGPLGLPGGGGSGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVP GHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPPHGVPGVGVPGHGVPGHGVP GHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVP GHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVP GHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVP GHGVPGHGGGGGSHHHHHH Example 2 This embodiment describes the expression and purification of the Ts-ELP fusion protein; BL21(DE3) stab strains were spread onto LB agar plates (Ampicillin) and cultured overnight at 37 ℃ and 200 rpm. Single colonies were picked and amplified in 5 mL LB medium until the logarithmic growth phase (OD600 nm ≈ 0.6-0.8), then transferred to 1 L LB medium and cultured at 37 ℃ and 200 rpm. After reaching the logarithmic growth phase, IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration 0.5 mM) was added, and the cells were cultured overnight at 20 ℃ and 200 rpm to induce protein expression. The cells were collected by centrifugation at 6000 rpm for 20 min, and then the bacteria were lysed using a cell sonicator to release the protein. After centrifugation at 12000 rpm for 30 min to remove bacterial debris, the target protein Thrombin-Ts-ELP was separated by Ni affinity chromatography. The target protein was replaced with Tris-NaCl (pH=8.0) buffer using a desalting column, followed by the addition of thrombin (200 U / mL) (protein:thrombin molar ratio = 1:5). The mixture was incubated overnight at 25 °C on a shaker at 100 rpm. The TrxA and thrombin peptide fragments were then removed from the fusion protein to obtain the complete Ts-ELP (targeted) or MTs-ELP (non-targeted) delivery vector. Further purification was then performed using a Ni affinity column and a desalting column to obtain high-purity target protein. The protein was then replaced with PBS buffer and stored at -80 °C for later use. Protein purity was determined using SDS-PAGE. The SDS-PAGE gel electrophoresis image and purification elution image of the purified fusion protein are shown below. Figure 2 As shown, Figure 2 In the figure, A and B represent the purification results of Ts-ELP and MTs-ELP proteins, respectively. The results show that recombinant proteins with a purity of >90% were successfully obtained through purification.

[0023] Example 3 This embodiment describes the preparation process of the self-assembled nano-antibiotic Tig@TsE; The Ts-ELP solution (0.5 mg / mL) was stirred at 100 rpm on a magnetic stirrer. Tigecycline (Tig) aqueous solution (4 mg / mL) was added dropwise, and stirring was continued for 30 min after mixing. The mixture was transferred to a water bath and incubated at 50°C for 5 min to encapsulate the Tig. After removal, zinc chloride (ZnCl2) was added and mixed well. ZnCl2 served as a stabilizer to maintain the formed nanostructure. The mixture was placed in an ultrafiltration centrifuge tube (3.5 kDa, Millipore, Massachusetts, USA) and centrifuged at 4000 g for 30 min. After washing with 10 volumes of PBS (10 mM, pH 7.4) and continuing ultrafiltration, the self-assembled nano-antibiotic Tig@Ts-ELP (Tig@TsE) was finally obtained. The control group, the non-targeted self-assembled nano-antibiotic Tig@MTs-ELP (Tig@MTsE), was obtained using the same method. A schematic diagram of the preparation of the nano-antibiotic Tig@TsE is shown below. Figure 11 As shown.

[0024] Example 4 In this embodiment, the particle size-light intensity of the nano-antibiotic Tig@TsE prepared in Example 3 was investigated at different pH values. Dynamic light scattering (DLS) was used to investigate the particle size-light intensity.

[0025] Particle size and polydispersity index (PDI) were determined by DLS at 633 nm using a Zetasizer Nano ZS (Malvern, UK). Samples were filtered through a 0.22 µm microporous membrane before analysis.

[0026] The results are as follows Figure 3 As shown, the DLS results indicate that under physiological conditions, Tig@TsE particles have a diameter of approximately 150 nm. After incubating Tig@TsE in different pH environments, it was observed that the particle size of Tig@TsE decreased as the pH value decreased, thus demonstrating that Tig@TsE possesses pH-responsive drug release characteristics.

[0027] Example 5 In this embodiment, the morphology of the proteins Ts-ELP and MTs-ELP prepared in Example 2 and the nano-antibiotics Tig@TsE and Tig@MTsE prepared in Example 3 were examined; the morphology of the fusion proteins was examined using transmission electron microscopy (TEM).

[0028] The prepared sample was dropped onto a copper grid, stained with a 2wt% phosphotungstic acid solution, and then placed on filter paper to air dry at room temperature. The morphology of the particles was observed and photographed using TEM.

[0029] The results are as follows Figure 4 As shown, where Figure 4 Images (a) and (c) in the image represent TEM images of Ts-ELP and MTs-ELP monomeric proteins, respectively. Figure 4 Images (b) and (d) in the image represent TEM images of Tig@TsE4 and Tig@MtsE, respectively. It can be seen that the particle sizes of both Ts-ELP and MTs-ELP observed by TEM are approximately 20 nm to 50 nm. Figure 4 (a) and (c) in the image), and both of them showed a significant increase in particle size after incubation with Tig, approximately 50 nm to 150 nm. Figure 4 (b) and (d) in the figure). The data show that Ts-ELP or MTs-ELP self-assembles with Tig after incubation to form nano-antibiotic drug delivery systems Tig@TsE4 and Tig@MtsE that encapsulate Tig.

[0030] Example 6 In this embodiment, the drug release capacity of the nano-antibiotic Tig@TsE prepared in Example 3 at different pH values ​​was examined, and the pH sensitivity and cumulative drug release of the low nano-antibiotic were analyzed. The in vitro drug release characteristics of the nano-antibiotic delivery system were evaluated using the dialysis bag method. 1 mL of Tig, Tig@TsE, and Tig@MTsE solutions (Tig concentration 2 mg / mL) were added to a dialysis bag (MW: 3.5 kDa) and placed in 100 mL of buffer solutions at different pH values ​​(pH 5.0, pH 6.5, pH 7.4). Dialysis was performed at 37 °C with constant shaking (100 rpm). 0.3 mL of the released fluid was collected at predetermined time points (0 h, 2 h, 4 h, 8 h, 24 h, 48 h), and an equal volume of fresh buffer was added simultaneously. The Tig concentration in the released fluid was determined by HPLC-MS (Waters 2707, USA), with D9-Tig used as an internal standard (IS). The collected samples were diluted 10-fold with a blank matrix, and then 0.3 mL of methanol (500 ng / mL) containing the internal standard was added. After vortexing for 3 min, the mixture was centrifuged at 2500 g for 12 min at 4°C. The supernatant was used for HPLC-MS determination of TIG concentration. Triples were performed for each measurement.

[0031] The results are as follows Figure 5As shown, at buffer pH=7.4, free Tig can be rapidly released in a short time, with a cumulative release of 80% within 4 hours, while Tig@TsE release is relatively slower, with a cumulative release of 20% within 48 hours. As the buffer pH decreases, the release rate of Tig@TsE increases significantly. At pH=6.5, the cumulative release of Tig reaches 60% within 8 hours, while the release rate is fastest at pH=5.0, with a cumulative release of 80% within 8 hours. This indicates that Tig@TsE can rapidly release drugs under acid-sensitive conditions.

[0032] Example 7 In this embodiment, the specific binding of Ts-ELP to endotoxin (LPS) is described.

[0033] Fluorescent probes Cy5.5-Ts-ELP or Cy5.5-MTs-ELP were obtained by labeling Ts-ELP or MTs-ELP with Cy5.5 (protein:Cy5.5 molar ratio = 1:5). These probes were then incubated with CRKP or KPN (ATCC700603) at 37 °C for 2 h or 6 h at a concentration of 4 μg / mL. After centrifugation and washing, the probes were resuspended in physiological saline, and the suspension was dropped onto a glass slide. The binding of the fluorescent probes to bacteria was observed under a laser confocal microscope.

[0034] result Figure 6 As shown, bacteria incubated with Cy5.5-Ts-ELP for 2 h exhibited strong fluorescence intensity for KPN. The fluorescence intensity increased in all groups as the incubation time was extended to 6 h. A completely consistent fluorescence intensity trend was observed in the CRKP group; this indicates that the Ts peptide, after fusion with ELP, still retains its LPS-targeting recognition function, and the binding to LPS on the bacterial surface is significantly time-dependent.

[0035] Example 8 In this embodiment, the in vitro antibacterial activity of Tig@TsE was verified.

[0036] Adjust the concentration of KPN (Klebsiella pneumoniae) or CRKP to 1 × 10⁻⁶. 6 CFU / mL was added to each well of a 96-well plate at a concentration of 100 μL. The concentrations of Tig and the fusion antimicrobial peptide were diluted to the MIC and added to each well at a concentration of 100 μL. The plates were incubated with bacteria at 37°C. The absorbance of the mixed bacterial solution at 600 nm was measured at predetermined time points (0 h, 2 h, 4 h, 6 h, 10 h, 12 h, 20 h, 24 h), and the corresponding bacterial growth curves were plotted. The experiment was repeated three times.

[0037] The results are as follows Figure 7As shown, Figure 7 In this context, A and B represent the MIC values ​​of Tig@TsE for KPN and CRKP, respectively. Figure 7 In the diagram, C and D represent the growth inhibitory effects of Tig@TsE on KPN and CRKP, respectively. It can be seen that for KPN, in the presence of the same concentration of MMP9 enzyme in the incubation system, Tig@TsE showed the lowest MIC value (0.25 μg / mL), lower than the MIC of Tig@MTsE (0.5 μg / mL), and also lower than the MIC values ​​of Tig and Ts alone (e.g., ...). Figure 7 (As shown in A). For CRKP, Tig@TsE had the lowest MIC value (2 μg / mL), lower than the MIC of untargeted modified Tig@MTsE (4 μg / mL), and also lower than the MIC values ​​of Tig or Ts administered alone (e.g., ...). Figure 7 (As shown in B). During the 24-hour incubation period, the Tig@TsE treatment group supplemented with MMP9 enzyme showed the lowest bacterial concentration, indicating the strongest bacterial growth inhibition. Figure 7 (As shown in C and D in the diagram). This demonstrates that the self-assembled nanodelivery system modified with Ts antimicrobial peptides can effectively encapsulate Tig. The precisely designed Ts-ELP can sensitively respond to high concentrations of MMP9 enzymes in the environment, fully exposing the Ts peptides to the infection site, exerting a synergistic antimicrobial effect with Tig, and ultimately enhancing the antimicrobial activity of Tig.

[0038] Example 9 In this embodiment, the induced drug resistance of self-assembled nano-antibiotics is analyzed; Adjust the concentration of KPN or CRKP to 0.5 McFarland (1 × 10⁻⁶) using MH medium. 8 A bacterial suspension (CFU / mL) was diluted 100-fold and 100 μL (1 × 10⁻⁶ CFU / mL) was added to each well of a 96-well plate. 5 CFU (Chemical Nucleotide Fuel) was used to adjust the concentration of nano-antibiotics Tig@TsE+MMP-9 or Tig@MTsE+MMP-9 (MMP-9: 0.25 μg / mL) to 1 / 2 MIC, MIC, 2 MIC, 4 MIC, and 8 MIC, respectively. These were then co-incubated with KPN and CRKP at 37 ℃ for 24 h (first generation). The absorbance was measured at 600 nm after incubation to determine the drug's MIC value. Bacteria grown in sub-MIC concentration wells were collected and cultured for 24 h using the same method to determine the second generation MIC value. This process was repeated for 7 generations, and the final MIC value was measured. The Tig-induced group treated in the same way served as the control group.

[0039] The results are as follows Figure 8As shown, at the endpoint of the assay, the MIC value of the Tig treatment group increased significantly to 8 μg / mL, which was 16 times higher than the MIC value of day 0 (0.5 μg / mL). The MIC value of Tig@MTsE increased from 0.5 μg / mL to 4 μg / mL, which was 8 times higher. Tig@TsE showed the weakest induction of drug resistance, with the MIC value increasing by only 2 times.

[0040] Example 10 In this embodiment, the in vivo biodistribution of self-assembled nano-antibiotics was analyzed.

[0041] Cy5.5 fluorescently labeled nanoantibiotics, Tig@TsE-Cy5.5 or Tig@MTsE-Cy5.5, were prepared. Five hours after BALB / c mouse modeling, mice were randomly assigned to groups and injected via tail vein with Free Cy5.5, Tig@TsE-Cy5.5, or Tig@MTsE-Cy5.5. Fluorescence images of Cy5.5 were acquired in a small animal in vivo imaging system at 5 and 24 hours after administration. Mice were euthanized by inhaling carbon dioxide after isoflurane anesthesia, and isolated organs were collected, along with Cy5.5 fluorescence images of the isolated organs. Parallel injections of Saline served as a negative control group.

[0042] The results are as follows Figure 9 As shown, Figure 9 In the figure, A represents near-infrared in vivo imaging of small animals 4 h and 24 h after tail vein administration of KPN to mice with lung infection. Figure 9 In the figure, B indicates near-infrared imaging of ex vivo organs 4 h and 24 h after KPN pneumonia tail vein injection. Figure 9 In the figure, C represents near-infrared in vivo imaging of small animals 4 h and 24 h after CRKP lung infection via tail vein administration. Figure 9 In the figure, D represents near-infrared imaging of ex vivo organs 4 h and 24 h after CRKP tail vein injection; it can be seen that the targeted nano-antibiotic Tig@TsE-Cy5.5 showed similar in vivo distribution characteristics in the KPN or CRKP groups, with targeted aggregation to the lungs observed 4 h after administration, while no lung aggregation was observed with free Cy5.5 or Tig@MTsE-Cy5.5. Figure 9 (A and C in the original text). In vitro tissue imaging showed that the fluorescence signal in the lungs of mice treated with Tig@TsE-Cy5.5 was significantly stronger than that in mice treated with free Cy5.5 or Tig@MTsE-Cy5.5, and the fluorescence intensity at 24 h was significantly stronger than that at 4 h. Figure 9 (B and D in the text).

[0043] Example 10 In this embodiment, an in vivo antibacterial experiment was conducted as follows: Five hours after BALB / c modeling, mice were randomly divided into groups and injected via tail vein with Tig, Ts, Ts-ELP, or the nano-antibiotic Tig@TsE or Tig@MTsE (at equivalent Tig or Ts-ELP concentrations), twice daily (12 hours / time), once in the morning and once in the evening, for a total of 5 days. Mice were weighed daily, and survival time was recorded. When the first mouse died, three mice from each group were randomly selected, and their alveoli were lavaged with pre-cooled PBS. The lavage fluid was collected and spread on blood agar plates to calculate the bacterial load in the lungs.

[0044] The results are as follows Figure 10 As shown, Figure 10 In this text, A and B represent the survival time of KPN and CRKP pneumonia mice after treatment with the drug via tail vein injection, respectively. Figure 10 In this context, C represents the bacterial load in the BALF of KPN or CRKP model mice; it can be seen that after KPN and CRKP infected mice were treated with different drugs, the survival time of mice treated with Tig@TsE was significantly prolonged. Figure 10 (A and B in the text). Bacterial load in the lungs after drug treatment was assessed by plate count. After treatment, BALF from the KPN and CRKP groups was diluted 10⁻⁶ times. 6 and 10 9 The blood was then evenly spread onto blood agar plates and incubated overnight at 37°C. In both models, the Tig@TsE treatment group showed the lowest BALF colony count, and the BALF bacterial load was significantly lower than in the other treatment groups. Figure 10 (C in the text). This demonstrates that in mouse models of acute pneumonia caused by KPN or CRKP infection, intravenous injection of Tig@TsE significantly reduced the bacterial load in the lungs of the model mice, thereby improving the mortality rate of CRKP-mediated lung infection and showing stronger antibacterial activity than Tig or Tig@MTsE.

[0045] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A Ts-ELP temperature-sensitive self-assembling nano-fusion protein, characterized in that, Comprise: antibacterial peptide Ts and elastin-like polypeptide ELP connected in turn; an MMP-9 enzyme cutting site is introduced between the antibacterial peptide Ts and the elastin-like polypeptide ELP.

2. The Ts-ELP temperature-sensitive self-assembling nano-fusion protein according to claim 1, wherein, The sequence of the elastin-like polypeptide ELP is (VPG(VH4)G)n, wherein n is a natural number of 3-100.

3. The Ts-ELP temperature-sensitive self-assembling nano-fusion protein according to claim 1, wherein, The MMP-9 enzyme cutting site is any amino acid sequence containing GPLGLPG.

4. The Ts-ELP temperature-sensitive self-assembling nano-fusion protein according to claim 1, wherein, The amino acid sequence of the fusion protein is shown as SEQ ID NO.

1.

5. A method of producing the Ts-ELP temperature-sensitive self-assembling nano fusion protein according to any one of claims 1 to 4, characterized in that, Comprise the following steps: antibacterial peptide Ts and elastin-like polypeptide ELP are genetically recombined by using DNA recombination technology to construct a recombinant plasmid; the constructed recombinant plasmid is transformed into competent cells BL21(DE3), and isopropyl-beta-D-thiogalactoside is used to induce the expression of the fusion protein; after the bacteria are lysed, protein purification is carried out; the purified protein is enzymatically cut by using thrombin; the protein after enzyme cutting is subjected to secondary purification to obtain the target fusion protein.

6. The use of the fusion protein of any one of claims 1-4 in the preparation of an antibacterial drug or a drug carrier for gram-negative bacillus infection.

7. Use according to claim 6, characterized in that, The gram-negative bacillus is CRKP or KPN.

8. A pharmaceutical carrier, characterized in that, Comprise the fusion protein of any one of claims 1-4.

9. A nanobiotic delivery system, characterized in that, Comprise the fusion protein of any one of claims 1-4, and tigecycline encapsulated on the fusion protein.

10. The method of claim 9, wherein the nanobiotic delivery system is prepared by, Comprise: the fusion protein is incubated with tigecycline at 50℃, and then ZnCl2 is added to self-assemble into a nanobiotic delivery system.

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