CN113648330A - Application of caffeic acid carbon dots in bacteriophage resistance - Google Patents
Application of caffeic acid carbon dots in bacteriophage resistance Download PDFInfo
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Abstract
The invention discloses an application of caffeic acid carbon dots in bacteriophage resistance, wherein the carbon dots prepared by using caffeic acid as a raw material are applied to the field of bacteriophage resistance, and the structure of bacteriophage protein is changed by utilizing the binding reaction of the caffeic acid carbon dots and the bacteriophage protein, so that the biological activity of the bacteriophage protein is lost.
Description
Technical Field
The invention relates to the field of virology and biotechnology, in particular to application of caffeic acid carbon dots in bacteriophage resistance.
Background
The virus is a small individual, has simple structure, no enzyme system and no metabolic mechanism, only contains a nucleic acid (DNA or RNA), and utilizes the substance and energy of living cells to complete self-replication and proliferation. Despite the benefits of recent viral research, such as the use of bacteriophages to treat some bacterial infections; the use of insect viruses is useful for the treatment and prevention of some diseases of agricultural pests, but the impact of viruses on human life remains enormous, such as Human Immunodeficiency Virus (HIV), Rabies Virus (RV), Hepatitis Virus (HVs), Norwalk Virus (NV), Ebola virus (EboV), and others. In recent years, antiviral drugs are not only various vaccines, but also nanotechnology is widely used in the development of antiviral drugs. For example, rapid advances in nanoscience and technology have provided potential alternatives to antiviral therapies, including metal nanoparticles, nanographene, and functional Carbon Dots (CDs). While nanotechnology has attracted extensive attention in many different fields, CDs have smaller size, larger surface area, lower toxicity, greater safety, and better biocompatibility than other nanomaterials. However, since new antiviral drug resistant strains are often developed in scientific research at present, there is a need to constantly search for or develop new antiviral compounds.
Disclosure of Invention
In order to solve the defects in the prior art, the inventor provides the application of caffeic acid carbon dots prepared from caffeic acid in bacteriophage resistance so as to meet the requirements of scientific research.
The caffeic acid carbon dots are prepared from caffeic acid serving as a raw material by a hydrothermal method, have obvious bacteriophage resistance, provide theoretical guidance for synthesis of new materials, and provide a new angle for research in the cross field of materials science and biology.
The caffeic acid carbon dots (CA-CDs) are prepared by a hydrothermal synthesis method, which comprises the following steps:
mixing Caffeic Acid (CA) with sterile water 1: 100 g, mL, then the mixed solution was transferred to a 100mL Teflon autoclave, reacted at 180 ℃ and 200 ℃ for 6h, cooled at room temperature, centrifuged (4500g, 15min), and filtered through a 0.22 μm filter membrane. The filtered solution was dried by a vacuum freeze dryer to obtain a dark red powder. Finally, the CA-CDs powder was stored at 4 ℃.
The invention utilizes the combination reaction of the caffeic acid carbon points and the bacteriophage protein to change the structure of the bacteriophage protein and lose the biological activity of the bacteriophage protein, and experiments show that the caffeic acid carbon points have the bacteriophage resistance which is not possessed by caffeic acid and is definite, and have unexpected technical effects.
Drawings
FIG. 1 is a diagram of PFU of three phages under three actions of sterile water, caffeic acid and caffeic acid carbon points in example 2 of the invention;
FIG. 2 is a transmission electron microscope image of three phages after normal state and caffeic acid carbon dot treatment in example 2 of the invention;
FIG. 3 is a plot of PFU at different times for the three phages in example 3;
FIG. 4 is a histogram of PFU of three phages in example 4 after treatment with different concentrations of CA-CDs solution;
FIG. 5 is a line graph of PFU of the three phages in example 4 after treatment with different concentrations of CA-CDs solution.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
Example 1 preparation of caffeic acid carbon dots (CA-CDs)
The method for preparing the caffeic acid carbon dots (CA-CDs) by using the hydrothermal synthesis method comprises the following specific steps:
mixing Caffeic Acid (CA) with sterile water 1: 100(g, mL), transferring the mixed solution to a 100mL polytetrafluoroethylene autoclave, reacting for 6h at the temperature of 180 ℃ and 200 ℃ (wherein the reaction is always under high pressure, the pressure of the 100mL polytetrafluoroethylene autoclave is usually not more than 3MPa), cooling at room temperature, centrifuging (4500g, 15min), and filtering with a 0.22 μm filter membrane. The filtered solution was dried by a vacuum freeze dryer (Duling commercial electric appliance Co., Ltd., China Hefei, Zhonghui) to obtain dark red powder. Finally, the CA-CDs powder was stored at 4 ℃.
Example 2 experiment of anti-phage Activity
Three phages, Siphoviridae (vB-Eos-IME167), Myoviridae (T4-phage), and Podoviridae (VMY22), were selected for anti-phage experiments:
(1) and (4) enriching and purifying the three phages, and storing by using corresponding host bacteria.
The host bacteria are used: the autoclaved tubes containing 5 ml of LB liquid medium were inoculated with vB-Eos-IME167 host, T4 phage host and VMY22 host, respectively. vB-Eos-IME167 host and T4 phage host were placed in a shaker at 37 deg.C and 150r/min, and VMY22 was placed in a shaker at 28 deg.C and 150r/min, and cultured until the density reached OD600 of 0.6-0.8.
Enrichment and purification of phage: transferring host bacteria to 100ml of LB liquid culture medium after autoclaving respectively according to 1% inoculum size, inoculating and culturing to OD600 of 0.6-0.8 respectively in corresponding shaking table, adding phage stock solution according to 10% inoculum size, continuing culturing, taking out and centrifuging (4500g, 15min), filtering with 0.22 μm filter membrane, and storing phage stock solution at 4 ° for later use after the liquid in the conical flask is clarified.
(2) CA-CDs and three phage incubation treatments.
The three phage stock solutions were diluted to gradient 10 using MM medium, respectively-1、10-2、10-3、10-4、10-5、10-6、10-7Standby; taking sterile water and caffeic acid monomer solution (2.5mg/mL) as a control group, CA-CDs (2.5mg/mL) as an experimental group, and mixing the sample and the diluted phage solution according to the proportion of 1: 2 (100. mu.L of sample + 200. mu.L of phage solution), vB-Eos-IME167 mix and T4 mix were incubated in a 37 ℃ incubator1h, culturing the VMY22 mixed solution in an incubator at 28 ℃ for 1 h.
(3) The three mixed solutions and the phage host bacterial liquid are mixed according to the proportion of 1: 2 incubation for 5 min.
Each 100. mu.L of the three mixtures was added to a sterilized EP tube containing 200. mu.L of the corresponding host bacterial culture, and incubated for 5min for a set of parallel experiments.
(4) And (3) by using a double-layer plate method, placing the sterilized LB semisolid culture medium containing the normal LB solid culture medium and 30% agarose in a microwave oven for heating and melting, transferring about 4mL of the solution incubated in the step (3) to mix when the temperature is cooled to 55-65 ℃, and pouring the mixed solution onto a plate containing the normal LB solid culture medium immediately after mixing.
(5) Culturing in an incubator.
The plates containing IME16 and the plates containing T4 were incubated at 37 ℃ in an incubator and the plates containing VMY22 were incubated at 28 ℃ in an incubator until plaques were completely developed, about 4-6 h.
(6) And (5) counting plaques.
Taking out the plate from the incubator, counting the plaque number of each plate, and calculating the plaque forming unit number (PFU), PFU/mL is plaque number multiplied by 10 phage dilution. Experimental data were processed using statistical analysis software.
As shown in fig. 1 (mean ± SD, n ═ 3, # p <0.05, # p <0.01, # p <0.001, # p <0.0001), sterile water and caffeic acid monomer solutions (2.5mg/mL) had similar Plaque Forming Units (PFU) for the three phages; the number of plaque forming units of CA-CDs (2.5mg/mL) was significantly different compared to sterile water and caffeic acid monomer solutions. CA-CDs can inhibit vB-Eos-IME167 cell proliferation by 1000 times, and inhibit T4 phage and VMY22 cell proliferation by more than 100 times.
The results of the double-plate method show that the CA-CDs can obviously reduce the activity of the phage. In order to judge the influence mode of CA-CDs on the activity of the phage more intuitively, the phage in a normal state and after the CA-CDs are treated are subjected to transmission electron microscope imaging respectively. As shown in FIG. 2, a and b are normal state and treated vB-Eos-IME167, c and d are normal state and treated T4 phage, respectively, and e and f are normal state and treated VMY22, respectively. From a and b, it can be seen that the treated vB-Eos-IME167 showed a curved tail and a rounded head compared to the normal state, indicating that the protein structure of vB-Eos-IME167 phage was altered, the biological activity was lost, and the host bacteria could not be normally infected. Through the observation of c and d, the head protein shell of the T4 phage is broken, namely the whole T4 phage morphology is destroyed, and the phage is inactivated by CA-CDs. In e, f, normal VMY22 was observed to be intact, whereas treated VMY22 was defective, shown by a reduction in the head protein shell and a deletion in the tail protein. As described above, the mechanism of the CA-CDs to resist phage can be attributed to the binding reaction of phage proteins with CA-CDs, which in turn changes their protein structure.
Example 3 treatment time experiment for phage Activity
Three phages, vB-Eos-IME167, T4-phage and VMY22, were selected for treatment time experiments.
(1) And (4) enriching and purifying the three phages, and storing by using corresponding host bacteria.
The host bacteria are used: the autoclaved tubes containing 5 ml of LB liquid medium were inoculated with vB-Eos-IME167 host, T4 phage host and VMY22 host, respectively. vB-Eos-IME167 host and T4 phage host were placed in a shaker at 37 deg.C and 150r/min, and VMY22 was placed in a shaker at 28 deg.C and 150r/min, and cultured until the density reached OD600 of 0.6-0.8.
Enrichment and purification of phage: transferring host bacteria to 100ml of LB liquid culture medium after autoclaving respectively according to 1% inoculum size, inoculating and culturing to OD600 of 0.6-0.8 respectively in corresponding shaking table, adding phage stock solution according to 10% inoculum size, continuing culturing, taking out and centrifuging (4500g, 15min), filtering with 0.22 μm filter membrane, and storing phage stock solution at 4 ° for later use after the liquid in the conical flask is clarified.
(2) Diluting the three phage stock solutions respectively by using MM culture media for later use; taking sterile water as a control group and CA-CDs solution (2.5mg/mL) as an experimental group, then adding 100 mu L of sample into 200 mu L of diluted phage, and respectively treating for different times (10, 30, 60, 120 and 180 min); the specific dilution of the phage is shown in the following table:
(3) each 100. mu.L of the three mixtures was added to a sterilized EP tube containing 200. mu.L of the corresponding host bacterial culture, and incubated for 5min for a set of parallel experiments.
(4) And (3) by using a double-layer plate method, placing the sterilized LB semisolid culture medium containing the normal LB solid culture medium and 30% agarose in a microwave oven for heating and melting, transferring about 4mL of the solution incubated in the step (3) to mix when the temperature is cooled to 55-65 ℃, and pouring the mixed solution onto a plate containing the normal LB solid culture medium immediately after mixing.
(5) The plates containing IME16 and the plates containing T4 were incubated at 37 ℃ in an incubator and the plates containing VMY22 were incubated at 28 ℃ until plaques were fully developed.
(6) The plates were removed from the incubator, and the number of plaques on each plate was counted separately, followed by calculating the number of Plaque Forming Units (PFU).
As shown in FIG. 3, a, d in FIG. 3 depict a 10 reduction in the infectious capacity of vB-Eos-IME167 over 180min5More than two times, and the CA-CDs are reduced by about 10min3And (4) doubling. FIG. 3 b, e shows the inhibitory effect of CA-CDs on T4 phage infection of host bacteria. The inhibition effect is 10 to 180min2Multiplied by 104Times, maximum PFU 1.4X 107Minimum PFU of 2.51X 105. Meanwhile, for VMY22, the plaque experiment showed that the antiviral ability of the experimental group was within the time-dependent range compared to the control group. As shown in c, f of FIG. 3, CA-CDs reduced the number of VMY22 from 10 to 10 times by plaque assay at treatment times from 10 to 180 minutes5More than twice. The result proves that the treatment time has an important influence on infecting host bacteria, and CA-CDs show stronger time-dependent antiviral activity between 10min and 180 min.
Example 4 concentration test against phage
Three phages, vB-Eos-IME167, T4-phage and VMY22, were selected for concentration experiments.
(1) And (4) enriching and purifying the three phages, and storing by using corresponding host bacteria.
The host bacteria are used: the autoclaved tubes containing 5 ml of LB liquid medium were inoculated with vB-Eos-IME167 host, T4 phage host and VMY22 host, respectively. vB-Eos-IME167 host and T4 phage host were placed in a shaker at 37 deg.C and 150r/min, and VMY22 was placed in a shaker at 28 deg.C and 150r/min, and cultured until the density reached OD600 of 0.6-0.8.
Enrichment and purification of phage: transferring host bacteria to 100ml of LB liquid culture medium after autoclaving respectively according to 1% inoculum size, inoculating and culturing to OD600 of 0.6-0.8 respectively in corresponding shaking table, adding phage stock solution according to 10% inoculum size, continuing culturing, taking out and centrifuging (4500g, 15min), filtering with 0.22 μm filter membrane, and storing phage stock solution at 4 ° for later use after the liquid in the conical flask is clarified.
(2) Diluting the three phage stock solutions respectively by using MM culture media for later use; taking sterile water as a control group as an experimental group, and then respectively adding 100 mu L of CA-CDs solutions (0.01, 0.1, 1, 5 and 10mg/mL) with different concentrations into 200 mu L of diluted phage for treatment for 60 minutes; the specific dilution of the phage is shown in the following table:
it should be noted that the significance of phage dilution is to ensure that the number of plaques on the double-layer plate is 300-100, which is convenient for the subsequent statistical counting and will not affect the experimental conclusion.
(3) Each 100. mu.L of the three mixtures was added to a sterilized EP tube containing 200. mu.L of the corresponding host bacterial culture, and incubated for 5min for a set of parallel experiments.
(4) And (3) by using a double-layer plate method, placing the sterilized LB semisolid culture medium containing the normal LB solid culture medium and 30% agarose in a microwave oven for heating and melting, transferring about 4mL of the solution incubated in the step (3) to mix when the temperature is cooled to 55-65 ℃, and pouring the mixed solution onto a plate containing the normal LB solid culture medium immediately after mixing.
(5) The plates containing IME16 and the plates containing T4 were incubated at 37 ℃ in an incubator and the plates containing VMY22 were incubated at 28 ℃ until plaques were fully developed.
(6) The plates were removed from the incubator, and the number of plaques on each plate was counted separately, followed by calculating the number of Plaque Forming Units (PFU).
As shown in FIGS. 4 and 5, the anti-phage ability increased with the increase of the concentration of CA-CDs. For the three phages, the capacity remained basically stable after the concentration of 5mg/mL, which indicates that the reaction of the phages with CA-CDs reached saturation, the antiviral effect of CA-CDs reached the maximum, and the anti-phage capacity did not increase after the concentration of 5 mg/mL.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (3)
1. Application of caffeic acid carbon dots in resisting bacteriophage is provided.
2. The use of caffeic acid carbon dots to combat bacteriophages according to claim 1, wherein the use of caffeic acid carbon dots to prepare bacteriophage resistant disinfecting articles.
3. The use of caffeic acid carbon dots to combat bacteriophages according to claim 1, wherein the caffeic acid carbon dots are made by:
mixing caffeic acid with sterile water according to the proportion of 1: 100(g, mL) to obtain a mixed solution;
transferring the mixed solution into a 100mL polytetrafluoroethylene autoclave, and reacting for 6h at the temperature of 180-;
then cooling at room temperature, and centrifuging for 15min under the condition that the centrifugal force is 4500 g;
centrifuging, and filtering with 0.22 μm filter membrane to obtain filtrate;
and drying the filtered solution by a vacuum freeze dryer to obtain the caffeic acid carbon points.
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| CN116656351A (en) * | 2023-05-15 | 2023-08-29 | 新乡医学院 | Preparation method and application of alcohol-soluble fluorescent carbon dots for detecting iron ions in organic phase |
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| JIRI KUDR等: "Carbon Dots Based FRET for the Detection of DNA Damage", 《BIOSENSORS AND BIOELECTRONICS》 * |
| PLINIO INNOCENZI等: "Carbon-based antiviral nanomaterials: graphene,C-dots, and fullerenes. A perspective", 《CHEM. SCI.》 * |
| TING DU等: "Carbon dots as inhibitors of virus by activation of type I interferon response", 《CARBON》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116656351A (en) * | 2023-05-15 | 2023-08-29 | 新乡医学院 | Preparation method and application of alcohol-soluble fluorescent carbon dots for detecting iron ions in organic phase |
| CN116656351B (en) * | 2023-05-15 | 2024-04-09 | 新乡医学院 | Preparation method and application of alcohol-soluble fluorescent carbon dots for detecting iron ions in organic phase |
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