CN116218773A - NK cell exosome of specific targeting bacteria and application thereof - Google Patents
NK cell exosome of specific targeting bacteria and application thereof Download PDFInfo
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- CN116218773A CN116218773A CN202310177189.0A CN202310177189A CN116218773A CN 116218773 A CN116218773 A CN 116218773A CN 202310177189 A CN202310177189 A CN 202310177189A CN 116218773 A CN116218773 A CN 116218773A
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Abstract
The invention discloses an Exosome (Exosome) secreted by natural killer cells (Natural killer cells, NK cells) activated by engineering bacteria, which has high-efficiency antibacterial performance and provides a new method for treating bacterial infection. The method comprises the following steps: (1) Unlike antibiotic therapy, NK cell-derived exosomes activated by engineered bacteria have bacterial targeting and lower toxic side effects, not being prone to drug resistance and allergic reactions; (2) The exosomes derived from NK cells activated by bacteria can be obtained through large-scale production, and the prepared exosomes can be stored at low temperature; (3) Has specific inhibition effect on bacteria and high targeting property.
Description
Technical Field
The present invention relates to the use of NK cell-derived exosomes (exosomes) activated by engineered bacteria in antibacterial applications.
Background
NK cells are the predominant effector cells in the innate immune system, and are involved not only in anti-tumor, anti-viral infections and immunomodulation, but also in the development of hypersensitivity reactions and autoimmune diseases in some cases. In addition, NK cells have the same 'immune memory' function as memory T cells and B cells, and can quickly start secondary immunity and exert stronger immune response when being contacted with the same antigen stimulation again. The surface of NK cell membrane expresses many receptors, and the activity of NK cells is regulated by different receptors so that the NK cells are in a silent or killing state. The activating receptors such as NKG2D can be obviously up-regulated after NK cells are stimulated by tumor cells, so that the killing function of the NK cells is enhanced. In addition to lysis of target cells by release of granzyme and perforin, NK cells trigger tumor cells to undergo apoptosis via the caspase pathway by stimulating death receptors on the surface of target cells. The present inventors believe that NK cells activate macrophages by releasing cytokines such as γ -IFN and eliminate bacteria by phagocytosis of macrophages. However, it is not clear whether NK cells are capable of direct killing of pathogens such as bacteria, and whether there are recognizing receptors on the surface of NK cells for bacteria has yet to be studied.
Exosomes, also known as exosomes, are nanoscale membranous vesicles secreted by cells and carrying cytoplasmic components, with a size of about 30-150 nm. Various cells of the body are capable of secreting such membranous vesicles, which are widely distributed in body fluids such as saliva, plasma, milk, etc. Exosomes were initially considered as "waste" of unwanted metabolism by cells, which were later found to contain large amounts of various bioactive substances such as proteins, RNAs, cytokines, etc. The exosomes transfer various contents including proteins, mRNA, miRNA and the like to other cells through membrane fusion, and serve as a bridge for cell-cell communication. The existing research shows that Antigen Presenting Cells (APC) and tumor cell derived exosomes exhibit strong immunostimulatory activity both in vivo and in vitro. Therefore, exosomes are being studied more and more. NK cells have strong anti-infection and tumor-clearing capacity, and the released NK cell exosomes contain a large amount of perforin and granzyme equivalent proteins, and can play an unknown and huge role in the synergic anti-tumor and antibacterial processes of the NK cells. Previous research work has found that NK exosomes induced to activate by engineered trophoblasts have antibacterial capacity, exhibit killing effects on a variety of bacteria, and have broad-spectrum antibacterial activity. However, partial bacteria play a beneficial role in human body, and the preparation of targeted NK cell exosome for directionally clearing pathogenic bacteria is important for maintaining the balance of human body flora.
With the development of chemical biotechnology, bacteria have become an emerging class of delivery systems for use in various biomedical research applications. Bacteria can be classified into gram-positive bacteria and gram-negative bacteria according to the cell wall structure and chemical composition of the bacteria. Gram-positive bacteria have thicker cell walls but relatively simple chemical compositions, containing about 90% peptidoglycan and about 10% teichoic acid. Gram-negative bacteria have a thin cell wall, a multi-layered structure (e.g., peptidoglycan and lipopolysaccharide layers), and contain certain amounts of lipids, proteins, and peptidoglycans. The cell wall components of bacteria contain various reactive sites (e.g., free sulfhydryl groups and amino groups) that are capable of reacting with various chemical molecules to effect surface modification of the bacteria. The combination of chemical biotechnology with bacteria provides great potential for engineering of bacteria and clinical applications based on bacterial delivery systems.
The clinical treatment of bacterial infections mainly relies on the use of antibiotics. Antibiotics commonly used in clinic can be classified into quinolones, beta-lactams (penicillins, cephalosporins, carbapenems, monocycles), macrolides, lincomycin, clindamycin, aminoglycosides, glycopeptides, oxazolidinones, metronidazole, tetracyclines, chloramphenicol, cyclic ester peptides, depending on the site targeted by the antibiotic. The use of antibiotics also results in the elimination of beneficial bacteria in the body while killing pathogenic bacteria. The widespread use of antibiotics has increased the frequency of microbial resistance and severe infectious diseases associated therewith at alarming rates over the past decades. The advent of multiple resistant bacteria has further exacerbated the non-drug availability of clinical patients. Thus, there is a great need for targeted antimicrobial agents in clinical therapy that can kill specific harmful pathogens without harm to the non-targeted flora.
Disclosure of Invention
The object of the present invention is to provide NK cell-derived exosomes (exosomes) activated by engineered bacteria and their use in antibacterial.
The invention aims at realizing the following technical scheme: engineering bacteria are constructed for stimulating and activating NK cells and secreting NK cell exosomes of targeted bacteria. Wherein the engineering bacterial membrane contains chemically coupled stimulating protein IL-21 and activating short peptide. Co-culturing the inactivated engineering bacteria and NK cells, controlling the concentration of IL-21 and activated short peptide in the culture solution to be 500IU/ml and 200ng/ml, and separating NK exosomes from the supernatant of the NK cell culture solution after at least 21 days of culture.
The activated short peptide sequence of the invention is as follows: IIDKSGAWV.
The invention also provides application of the exosomes in antibiosis, and the adopted engineering bacteria strain and the obtained exosome specific targeted strain are the same. Including but not limited to: bacillus tuberculosis, bacillus alcaligenes, bacillus amyloliquefaciens, bacillus brevis, bacillus circulans, bacillus clausii, bacillus coagulans, bacillus lautus, bacillus lentus, bacillus licheniformis, bacillus megaterium, bacillus stearothermophilus, bacillus subtilis, bacillus thuringiensis, corynebacterium, streptomyces lividans, streptomyces murinus, streptococcus pyogenes, streptococcus intermedius, streptococcus iniae, streptococcus pneumoniae, staphylococcus aureus, staphylococcus intermedia, staphylococcus pseudointermedia, staphylococcus epidermidis, enterococcus faecium, salmonella typhi, escherichia coli, vibrio cholerae, neisseria meningitidis, neisseria gonorrhoeae.
Further, the above application is: is used for preparing antibacterial preparation.
Further, the inactivation method of the invention includes, but is not limited to, irradiation inactivation, which can be performed on engineering bacteria by using a gamma-ray irradiator, for example, BIOBEAM GM2000, according to the radiation intensity of 10-50 Gy.
The invention has the beneficial effects that: the invention tests the exosome secreted by NK cells activated by engineering bacteria, finds that the exosome has remarkable targeted antibacterial capability, can be used for treating clinical drug-resistant bacteria, and comprises the following antibacterial means: (1) Unlike antibiotic therapy, NK cell-derived exosomes activated by engineered bacteria have bacterial targeting and lower toxic side effects, not being prone to drug resistance and allergic reactions; (2) The exosomes derived from NK cells activated by bacteria can be obtained through large-scale production, and the prepared exosomes can be stored at low temperature; (3) has extremely high targeted killing capability on bacteria.
Drawings
FIG. 1 is a Western blot assay;
FIG. 2 is a schematic diagram of value added activity; wherein A is the schematic diagram of proliferation activity of the irradiation inactivated trophoblast system; b is a schematic diagram of proliferation activity of radiation-inactivated engineering staphylococcus aureus;
FIG. 3 shows the differences in performance of the PBS control, the blank bacteria, the trophoblast system, and the engineered bacterial system to amplify NK;
FIG. 4 shows the expression of NK cell surface protein NKG2D activated by PBS control, blank bacteria, trophoblast system and engineered bacterial system;
FIG. 5 shows the expression of NK cell surface protein KIR2DS4 activated by PBS control, blank bacteria, trophoblast system, and engineered bacterial system;
FIG. 6 is a graph showing NK cell exocrine expression profile activated by an engineered bacterial system, wherein A is a particle size distribution map, B is a transmission electron microscope scan, and C is a protein differential expression analysis of the contents;
FIG. 7 is an identification of the ability of exosomes to inhibit bacterial proliferation by different drug resistance mechanisms;
FIG. 8 shows the in vivo bacteriostatic effect of NK exosomes activated by the engineered bacterial system, wherein A is the degree of auditory restitution in mice; b is a photograph of the eardrum membrane of the mouse, showing the recovery degree of the eardrum membrane;
FIG. 9 shows the antibacterial property of NK cell exosomes derived from Staphylococcus aureus.
FIG. 10 shows antibacterial property detection of NK cell exosomes derived from Acinetobacter baumannii.
FIG. 11 shows the antibacterial property detection of NK cell exosomes derived from Serratia.
Detailed Description
In order to further describe the technical means and effects adopted by the present invention for achieving the intended purpose, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present invention with reference to the accompanying drawings and preferred embodiments.
The invention is further illustrated below with reference to examples.
In order to verify the targeted antibacterial effect of the invention, the invention firstly constructs drug-resistant pathogenic bacteria as follows.
Collecting samples such as sputum, urine, secretion, blood, pus, lavage liquid, drainage liquid, catheter liquid, bile, ascites, cerebrospinal fluid, hydrothorax, puncture liquid/substance, swab, tissue block, exudate/liquid, bone marrow, joint fluid, stool, leucorrhea and the like which are sent out by clinical departments. All specimens are strictly collected according to the detection requirement, repeated strains separated from the same part of the same patient are removed, and the strains are identified. Sputum requires sputum smear to evaluate the quality of the specimen, and only smear confirmed to be a qualified lower respiratory tract isolate and cultured for more than three positives can be included in the study. Bacterial culture and identification were performed according to the national clinical test protocol. The quality control strain is staphylococcus aureus, streptococcus pneumoniae, enterococcus faecalis, pseudomonas aeruginosa, ESBL+ strain, klebsiella pneumoniae and haemophilus influenzae.
Bacterial resistance monitoring was tested using standard antibacterial drug paper spreading methods and full-automatic bacterial identification drug sensitive analyzers, including ampicillin, cefazolin, ciprofloxacin, levofloxacin, cefuroxime, sulfamethoxazole, ceftriaxone, ampicillin and sulbactam, aztreonam, cefepime, doxycycline, ceftazidime, amoxicillin and clavulanic acid, minocycline, tobramycin, ticarcillin and clavulanic acid, cefoperazone and sulbactam, gentamicin, piperacillin and tazobactam, amikacin, ceftioxime, tetracycline, cefuroxime, cefpodoxime, imipenem, meropenem, cefotetan, moxifloxacin, ceftizoxime, nitrone, chloramphenicol, ertapenem, tigecycline, polymyxin B, and the like.
As shown in the following table, we isolated and obtained chloramphenicol, lincomycin, ofloxacin and other drugs such as Staphylococcus aureus, pseudomonas aeruginosa, streptococcus agalactiae, candida parapsilosis, acinetobacter baumannii, serratia.
Example 1
Step 1: construction and characterization of engineered staphylococcus aureus.
IL-21 and an artificially synthesized activated short peptide (sequence IIDKSGAWV) were dissolved in BES buffer at 500IU/ml and 200ng/ml, and 1X 10-A6 Staphylococcus aureus ATCC6538 was added to BES buffer and reacted at 37℃for half an hour. IL-21 and the activated short peptide were coupled to the surface of Staphylococcus aureus using a succinimidyl ester-amino coupling method. The reaction reagent and unconjugated short peptide were removed by multiple centrifugation washes. As shown in FIG. 1, two specific bands appear in the protein expression of engineered Staphylococcus aureus, which are sized to match the molecular mass of IL-21 and the activated short peptide, as compared to unmodified Staphylococcus aureus.
Step 2: inactivation of trophoblasts and engineered staphylococcus aureus.
Taking the prepared engineering staphylococcus aureus sample and K562 trophoblast which is independently constructed in a laboratory, and respectively carrying out the steps of 60 Co-gamma rays are irradiated at a dose of 20kGy, the morphology of cells and staphylococcus aureus is observed through a microscope, and the activity of trophoblasts and engineered staphylococcus aureus is detected through a CCK-8 detection kit and an optical density absorption method. As shown in fig. 2 a and B, the irradiated trophoblasts and engineered staphylococcus aureus lost proliferation activity.
Step 3: the feeder cell system and the engineered staphylococcus aureus system amplify NK cells.
50mL of peripheral blood was collected intravenously, diluted with PBS buffer, and PBMC cells were obtained using Ficoll lymphocyte isolate. NK cells were labeled with CD56 magnetic beads and then subjected to cell sorting. The sorted cells were according to 1X10 6 the/mL was resuspended in NK cell medium. And respectively evaluating the amplification effect of the trophoblast system and the engineered staphylococcus aureus system on NK cells. The control group and the experimental group were set as follows:
a first group: PBS control group;
second group: k562 feeder cells;
third group: blank staphylococcus aureus;
fourth group: engineering staphylococcus aureus modified in step 2.
Four groups of amplified vectors and NK cells were cultured at 37℃and 5% CO, respectively 2 In an incubator. NK cells were counted on alternate days. As shown in fig. 3, after 21 days of expansion, the negative control group (PBS control group and blank staphylococcus aureus group) could not expand NK cells, the positive control group (K562 trophoblast) had about 4000 times of NK cells expanded, and the engineered staphylococcus aureus system had more than 3000 times of NK cells expanded, indicating that the engineered staphylococcus aureus system could also effectively induce NK cell expansion.
Step 4: detecting the expression of proteins on the surface of NK cells;
NK cells cultured for 21 days in the above examples were collected, and after labeling the NK cells with CD56 magnetic beads, cell sorting was performed, and the purity of NK cells detected by flow cytometry was 95% or more. Take 1x10 6 NK cells were analyzed for NK cell expression protein levels using specific flow antibodies CD56, NKG2D, KIR DS4. As shown in fig. 4 and 5, the trophoblast system (second group) and the engineered staphylococcus aureus system (fourth group) were able to up-regulate the expression of NK cell-activating receptor NKG2D compared to PBS control and blank staphylococcus aureus, while the engineered bacterial system was able to further up-regulate KIR2DS4 receptor targeted to killer bacteria.
Step 5: analysis of NK cell exosome content.
NK cell culture solution cultured for 21 days in the above example was collected, cell pellet was removed by centrifugation at 400g for 5min in a table type low speed centrifuge, and supernatant was collected for 4℃to be preserved for use. The exosomes in the culture broth were purified using a hollow fiber tangential filtration system (Spectrum Laboratories KrosFlo Research II TFF System). Firstly, removing cell debris in a cell culture solution by using a 0.45 mu m mPES hollow fiber filter column (P-S02-E45U-10-N); the filtrate is further concentrated by a mPES hollow fiber filter column (S02-E300-05-N) with the molecular weight cut-off of 300-kDa to obtain an exosome crude product; to further reduce the volume and remove residual media and salt ions, the crude exosome preparation was diluted with 3 volumes of PBS and concentrated using a mPES hollow fiber filter column (D02-E300-05-N) with a molecular weight cut-off of 300-kDa to give the exosome of very high purity.
Resuspension the obtained exosomes with deionized water, placing a small amount of exosomes in a copper mesh with a carbon coating, removing redundant moisture, dyeing with 2% tungsten phosphate, naturally drying, and observing the morphology and the size of the exosomes by using a transmission electron microscope. As shown in A and B of FIG. 6, NK cell exosomes are about 200nm in size, and TEM results show that isolated NK exosomes have typical exosome structures and present closed membrane structures with semitransparent, elliptic and different sizes inside.
Protein quantification is carried out on exosomes by adopting a BCA protein quantification kit, proteins in the exosomes are released by using a lysate, and the exosome Marker protein CD63 and NK exosome characteristic protein NKG2D are detected by adopting a Werstern blot method. As shown in FIG. 6C, exosomes released by NK cells expanded by the engineered bacteria contain the cytotoxic receptor KIR2DS4 specific for killer bacteria as compared to NK cells expanded by trophoblasts.
Step 6: testing the inhibition ability of exosomes to the proliferation of staphylococcus aureus of different samples;
samples of staphylococcus aureus 2020111301, 2020112001-1, 2020112503, 2020112701-2 and 2020112702-1 were taken, the NK exosomes obtained in step 5 were mixed with the 5 samples of staphylococcus aureus in different proportions, and the number of bacteria was quantified by measuring Optical Density (OD). The results are shown in figure 7, NK cell exosomes exhibit killing activity at lower concentrations (10 ug/ml) i.e. against all staphylococcus aureus samples. It can be seen that exosomes induced by engineering bacteria of staphylococcus aureus exhibit excellent killing power against all staphylococcus aureus.
Step 7: testing in-vivo antibacterial effect of exosomes;
in order to determine the in vivo bacteriostatic activity of exosomes, we selected staphylococcus aureus to model mice and treated the model mice with NK exosomes obtained in step 5 at different doses, and evaluated the treatment by evaluating the mouse hearing threshold. The results are shown in fig. 8 a and B, and the auditory threshold of the model mice treated with exosomes was restored to normal, compared to the negative control without exosomes, indicating that exosomes also had good effects in vivo.
Step 8: inhibition ability of exosomes against bacterial proliferation of different drug resistance mechanisms;
to examine whether the exosomes obtained by engineering amplification have the advantage of targeting bacteria and the ability to inhibit the growth of drug-resistant bacteria, we selected staphylococcus aureus (sample number 2020112502-2), pseudomonas aeruginosa (sample number 2020112001-2), streptococcus agalactiae (sample number 2020112502-1), candida parapsilosis (sample number 2020112502-2) as the detection subjects. The NK exosomes obtained in step 5 were mixed with the above 6 bacteria in different proportions and the number of bacteria was quantified by measuring the Optical Density (OD). The results are shown in FIG. 9, where NK cell exosomes exhibit killing activity against multi-resistant Staphylococcus aureus at lower concentrations (10 ug/ml) than negative controls without exosomes, but not significantly against several other common resistant bacteria. The results show that NK cell exosomes can achieve targeted killing by relying on a specific engineering bacterial amplification system and are not affected by bacterial drug resistance.
Example 2
Step 1: construction and characterization of engineering Acinetobacter baumannii.
IL-21 and an artificially synthesized activated short peptide (sequence IIDKSGAWV) were dissolved in BES buffer at 500IU/ml and 200ng/ml, and 1X 10-A6A. Baumannii SHBCC D10901B2471 was added to BES buffer and reacted at 37℃for half an hour. IL-21 and the activated short peptide are coupled to the surface of Acinetobacter baumannii by utilizing a succinimidyl ester-amino coupling method. The reaction reagent and unconjugated short peptide were removed by multiple centrifugation washes.
Step 2: inactivation of engineered acinetobacter baumannii.
Taking the prepared engineering Acinetobacter baumannii sample, and passing 60 Co-gamma rays are irradiated to kill the animals at a dosage of 20 kGy.
Step 3: and amplifying NK cells by an engineering Acinetobacter baumannii system to obtain exosomes.
50mL of peripheral blood was collected intravenously, diluted with PBS buffer, and PBMC cells were obtained using Ficoll lymphocyte isolate. NK cells were labeled with CD56 magnetic beads and then subjected to cell sorting. The sorted cells were according to 1X10 6 the/mL was resuspended in NK cell medium.
Culturing inactivated engineering Acinetobacter baumannii and NK cells at 37 ℃ with 5% CO 2 After 21 days of expansion culture in an incubator, NK cell culture fluid is collected, cell sediment is removed by centrifugation of 400g for 5min in a table low-speed centrifuge, and supernatant is collected and stored at 4 ℃ for later use. The exosomes in the culture broth were purified using a hollow fiber tangential filtration system (Spectrum Laboratories KrosFlo Research II TFF System). Firstly, removing cell debris in a cell culture solution by using a 0.45 mu m mPES hollow fiber filter column (P-S02-E45U-10-N); the filtrate is further concentrated by a mPES hollow fiber filter column (S02-E300-05-N) with the molecular weight cut-off of 300-kDa to obtain an exosome crude product; to further reduce the volume and remove residual media and salt ions, the crude exosome preparation was diluted with 3 volumes of PBS and concentrated using a mPES hollow fiber filter column (D02-E300-05-N) with a molecular weight cut-off of 300-kDa to give the exosome of very high purity.
Step 4: inhibition ability of exosomes against bacterial proliferation of different drug resistance mechanisms;
in order to examine whether the exosomes obtained by engineering amplification have the advantage of targeting bacteria and the ability to inhibit the growth of drug-resistant bacteria, we selected Acinetobacter baumannii (sample number 2020112701-1), pseudomonas aeruginosa (sample number 2020112001-2), streptococcus agalactiae (sample number 2020112502-1), and Candida parapsilosis (sample number 2020112502-2) as detection targets. The NK exosomes obtained in step 5 were mixed with the above 6 bacteria in different proportions and the number of bacteria was quantified by measuring the Optical Density (OD). As shown in fig. 10, NK cell exosomes showed killing activity against multi-resistant acinetobacter baumannii at lower concentrations (10 ug/ml) than negative controls without exosomes, but the killing effect against several other common resistant bacteria was not obvious. The results show that NK cell exosomes can achieve targeted killing depending on a specific engineered bacterial amplification system and are not affected by bacterial resistance.
Example 3
Step 1: construction and characterization of engineered Serratia.
IL-21 and an artificially synthesized activated short peptide (sequence IIDKSGAWV) were dissolved in BES buffer at 500IU/ml and 200ng/ml, and 1X 10-A6 Serratia SHBCC D12819 was added to BES buffer and reacted at 37℃for half an hour. IL-21 and the activated short peptide are coupled to the surface of Acinetobacter baumannii by utilizing a succinimidyl ester-amino coupling method. The reaction reagent and unconjugated short peptide were removed by multiple centrifugation washes.
Step 2: inactivation of engineered Serratia bacteria.
Taking the prepared engineering Serratia sample, and passing 60 Co-gamma rays are irradiated to kill the animals at a dosage of 20 kGy.
Step 3: and amplifying NK cells by an engineering Serratia system to obtain exosomes.
50mL of peripheral blood was collected intravenously, diluted with PBS buffer, and PBMC cells were obtained using Ficoll lymphocyte isolate. NK cells were labeled with CD56 magnetic beads and then subjected to cell sorting. The sorted cells were according to 1X10 6 the/mL was resuspended in NK cell medium.
Culturing inactivated engineering Serratia bacteria and NK cells at 37deg.C with 5% CO 2 After 21 days of expansion culture in an incubator, NK cell culture fluid is collected, cell sediment is removed by centrifugation of 400g for 5min in a table low-speed centrifuge, and supernatant is collected and stored at 4 ℃ for later use. The exosomes in the culture broth were purified using a hollow fiber tangential filtration system (Spectrum Laboratories KrosFlo Research II TFF System). Firstly, removing cell debris in a cell culture solution by using a 0.45 mu m mPES hollow fiber filter column (P-S02-E45U-10-N); the filtrate is further concentrated by a mPES hollow fiber filter column (S02-E300-05-N) with the molecular weight cut-off of 300-kDa to obtain an exosome crude product; to further reduce the volume and remove residual media and salt ions, the crude exosome preparation was diluted with 3 volumes of PBS and concentrated using a mPES hollow fiber filter column (D02-E300-05-N) with a molecular weight cut-off of 300-kDa to give the exosome of very high purity.
Step 4: inhibition ability of exosomes against bacterial proliferation of different drug resistance mechanisms;
to examine whether the exosomes obtained by engineering amplification have the advantage of targeting bacteria and the ability to inhibit the growth of drug-resistant bacteria, we selected Serratia (sample No. 2020112501), pseudomonas aeruginosa (sample No. 2020112001-2), streptococcus agalactiae (sample No. 2020112502-1), and Candida parapsilosis (sample No. 2020112502-2) as the detection subjects. The NK exosomes obtained in step 5 were mixed with the above 6 bacteria in different proportions and the number of bacteria was quantified by measuring the Optical Density (OD). The results are shown in FIG. 11, where NK cell exosomes exhibit killing activity against multidrug-resistant Serratia at lower concentrations (10 ug/ml) than negative controls without exosomes, but killing effect against several other common drug-resistant bacteria is not obvious. The results show that NK cell exosomes can achieve targeted killing by relying on a specific engineering bacterial amplification system and are not affected by bacterial drug resistance.
The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.
Claims (3)
1. A specific bacterial-targeted NK cell exosome, characterized by being obtained by the following method: inactivating engineering bacteria chemically grafted with IL-21 and activated short peptide on cell walls, co-culturing the engineering bacteria with NK cells for at least 21 days, and separating NK exosomes from an NK cell culture solution supernatant; wherein the engineering bacteria and the exosome targeted bacteria belong to the same type, and the sequence of the activated short peptide is shown as SEQ ID NO. 1; and the concentration of IL-21 and the activated short peptide in the culture solution of NK cells is 500IU/ml and 200ng/ml respectively.
2. The exosome of claim 1, wherein the engineered bacterium is one of a bacillus tuberculosis, bacillus alcalophilus, bacillus amyloliquefaciens, bacillus brevis, bacillus circulans, bacillus clausii, bacillus coagulans, bacillus lautus, bacillus lentus, bacillus licheniformis, bacillus megaterium, bacillus stearothermophilus, bacillus subtilis, bacillus thuringiensis, corynebacteria, streptomyces plumbiferum, streptomyces murinus, streptococcus pyogenes, streptococcus intermedium, streptococcus iniae, streptococcus pneumoniae, staphylococcus aureus, staphylococcus intermedia, staphylococcus pseudointermedia, staphylococcus epidermidis, enterococcus faecium, salmonella typhi, escherichia coli, vibrio cholerae, neisseria meningitidis, neisseria gonorrhoeae, and pseudomonas aeruginosa.
3. Use of an exosome according to claim 1 for the preparation of a specific targeted antibacterial formulation.
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