CN112611742B - Zika virus visual marking strategy utilizing photo-click bioorthogonal reaction - Google Patents
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Abstract
The invention discloses a Zika virus visual marking strategy utilizing optical click bioorthogonal reaction, which comprises the following steps: first functionalizing PQ into a ZIKV virus; functionalizing a vinyl ether into the quantum dots; ZIKV viruses were then labeled as fluorescent quantum dots by cyclization under irradiation. The invention has the advantages that: the quantum dots based on the photon-click bioorthogonal reaction are used for marking and tracking the ZIKV, so that the ZIKV has accurate time-space control visualization in the natural environment of the ZIKV. ZIKV-quantum dots are able to delineate the interaction of ZIKV with host cells under TPZ or norconazole treatment conditions, and thus the present invention provides a reliable tool for elucidating viral interactions with host cells and developing potentially rapid diagnostic and therapeutic methods.
Description
Technical Field
The invention relates to the technical field of biological small molecule markers, in particular to a Zika virus visual marker technology.
Background
Zika Virus mosquito (Zika Virus, ZIKV), belonging to the flaviviridae family of flaviviridae, is a re-emerging flavivirus that can cause various Zika syndromes, such as ventricular hypertrophy, by biting by infected mosquitoes or transmission by sexual contact with infected partners, and ZIKV infection during pregnancy is associated with many fetal malformations, including prenatal death and microcephaly. Recently, zika virus has been reported to outbreak in more than 30 countries or regions, and has become a major threat to global health. ZIKV has been studied relatively rarely, and thus the biology and pathogenesis of ZIKV infection is not fully understood. The general public in endemic/endemic countries and travelers to these areas urgently need an effective vaccine to inhibit transmission.
Currently, the diagnosis technology of ZIKV is mainly based on a reverse transcription polymerase chain reaction (RT-PCR) of serum or detection of a ZIKV specific IgM antibody, and the diagnosis technology makes remarkable progress in the aspect of preventing disease transmission. In addition, recent studies on ZIKV-infected cell signaling pathways have greatly enriched studies on RNA virus infection mechanisms. However, the visual studies of the interaction of Zika virus with host cells are still rare, and this is crucial for understanding the molecular mechanisms and pathogenesis of Zika virus disease. Therefore, the development of a fluorescence labeling method without antibody cross reaction is urgent for the elucidation of pathogenic mechanisms of viruses.
In order to reveal the interaction between viruses and host cells, fluorescent dyes are widely applied to virus labeling experiments and real-time imaging, and the understanding of people on the virus infection process is deepened. Photobleaching and spectral overlap of fluorophores is inevitable, however, greatly affecting the effectiveness of tracking dye-labeled viruses, limiting their use in biological imaging. Fluorescent quantum dots can be reasonably selected as candidate materials due to their unique optical properties, such as narrow-band, tunable fluorescence emission, high fluorescent quantum yield and photostability. Although many studies to construct quantum dot virus imaging modalities have been able to provide meaningful information, maintaining virus penetration after riveting viruses onto quantum dots by non-mild and uncontrollable physicochemical processes remains a huge challenge, and thus it is difficult to reveal true virus-host cell interactions by this approach.
Disclosure of Invention
The invention aims to provide a Zika virus visual marking strategy utilizing a light click bioorthogonal reaction, so as to solve the problem that the prior art is lack of a material basis for researching interaction of ZIKV viruses and host cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
a Zika virus visual marking strategy utilizing a light click bioorthogonal reaction comprises the following steps: functionalizing 9, 10-phenanthrenequinone into a ZIKV virus; functionalizing a vinyl ether into the quantum dots; the ZIKV virus was then labeled as fluorescent quantum dots by cyclization under irradiation.
Further, the air conditioner is provided with a fan,
the operation of functionalizing 9, 10-phenanthrenequinone into the ZIKV virus includes: dispersing 9, 10-phenanthrenequinone in MES buffer solution, adding EDC and NHS, performing ultrasonic action, sealing, and oscillating; dispersing the ZIKV virus treated in the step in PBS; MES buffer was added, shaken overnight, and purified by NAP-5 desalting column to obtain the Zuka virus modified with PQ group.
Further, the air conditioner is characterized in that,
the detailed procedures for functionalizing 9, 10-phenanthrenequinone into ZIKV viruses include: 11mg, 4.17X10 -5 mol of 9, 10-Phenanthrenequinone (PQ) dispersed in 10ml of MES buffer; 80mg of the active ingredient is added, and 4.17x10 is added -4 Performing ultrasonic treatment on mol EDC and 120mg NHS for 15s, sealing, and oscillating for 15min at 37 ℃; then, 100. Mu.L of Zika virus at a concentration of 2mg/ml was dispersed in 10ml of PBS, MES buffer was added thereto, the mixture was shaken overnight at 37 ℃ and purified by NAP-5 desalting column to obtain a Zika virus modified with PQ group.
Further, the air conditioner is provided with a fan,
the operation of functionalizing the vinyl ether into the quantum dots comprises: taking amino-containing quantum dots, taking carboxyl-containing vinyl ether, EDCI and 4-dimethylamino pyridine, respectively putting into a 3-neck flask, and adding dichloromethane to completely dissolve; then shaking in a dark environment at room temperature; and then carrying out silica gel column chromatography to obtain the quantum dot modified by the vinyl ether group.
Further, the air conditioner is provided with a fan,
the operation of functionalizing the vinyl ether into the quantum dots comprises: taking the molar concentration as 8 multiplied by 10 -6 Mu M quantum dot containing amino 1 mu L, then taking vinyl ether containing carboxyl 1000 mu L, EDCI 15.3mg and 4-dimethylamino pyridine 9.8mg, respectively putting into a 3-neck flask, adding 5ml dichloromethane and completely dissolving; then shaking for 2 hours in a dark environment at room temperature; and then carrying out silica gel column chromatography to obtain the quantum dot modified by the vinyl ether group.
Further, the air conditioner is provided with a fan,
the operation of labeling the ZIKV virus as fluorescent quantum dots by cyclization under irradiation comprises:
dissolving the 9, 10-phenanthrenequinone-group-modified Zika virus and the vinyl ether group-modified quantum dots in a mixed solution of CH3CN and PBS, and then irradiating the mixed solution for 1min by using an LED lamp to perform cyclization reaction to mark the ZIKV virus as fluorescent quantum dots, thereby obtaining the quantum dot-modified Zika virus ZIKV-Qds.
The advantages of the invention include: the ZIKV is labeled and tracked using quantum dots based on the light-click bioorthogonal reaction. It is possible to visualize ZIKV with precise temporal and spatial control in its natural environment. Using this strategy, ZIKV was successfully tracked and visualized after cell entry into different cell lines (e.g., a549 and SNP 19). Furthermore, ZIKV-quantum dots are able to delineate the interaction of ZIKV with host cells under TPZ or norconazole treatment conditions. The invention will provide a reliable tool for elucidating the interaction of viruses with host cells and for the development of potentially rapid diagnostic and therapeutic methods.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:
FIG. 1 is a schematic diagram of the synthesis process of ZIKV-QDs based on optical pointing;
FIG. 2 is a schematic diagram showing the change in absorption peaks during the light click reaction between PQ and an electron-rich vinyl ether;
FIG. 3 is a graph of experimental color changes of a light click reaction solution between PQ and an electron-rich vinyl ether;
FIG. 4 is an experimental graph of a ZIKV-quantum dot and quantum dot polyacrylamide gel electrophoresis method;
FIG. 5 is a ZIKV-Quantum dot Transmission Electron microscope image;
FIG. 6 is an isobologram of titers after purification;
FIG. 7 is a statistical plot of ZIKV RNA levels;
FIG. 8 is a homograph of NTA concentration and average particle size for ZIKV;
FIG. 9 is a statistical graph of the average particle diameters of nanoparticles and quantum dot-modified nanoparticles
FIG. 10 is a Zeta potential statistical plot for quantum dots and ZIKV viruses;
FIG. 11 is a ZIKV and ZIKV-quantum dot virus titer statistics plot;
FIG. 12 is a photograph of fluorescence images of A549 cells incubated with ZIKV-QDs and not incubated with ZIKV;
FIG. 13 is the co-localized scatter plot of FIG. 12;
FIG. 14 is the corresponding Pearson correlation coefficient (PC) of FIG. 12;
FIG. 15 is a photograph of fluorescent images of SNB 19 cancer cells incubated with ZIKV-QDs incubated with non-incubated ZIKV;
FIG. 16 is the co-localized scatter plot of FIG. 15;
fig. 17 is a Pearson correlation coefficient (PC) for the graph of fig. 15. (ii) a
FIG. 18 is a graph of the cellular fluorescence intensity experiment for the quantum dots of the control group, CPZ-treated group, and noridazole-treated group;
FIG. 19 is a graph showing the results of Western blotts on CPZ treatment;
FIG. 20 is a cell viability histogram of CPZ treatment;
FIG. 21 is a cell viability histogram of noridazol treatment;
fig. 22 and 23 are cell viability statistical graphs after incubation of quantum dots with different concentrations;
FIG. 24 is a statistical chart of the effect on cells of different irradiation times.
Detailed Description
The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.
The synthesis process of the ZIKV-QDs based on photo-click is shown in FIG. 1, and 9, 10-Phenanthrenequinone (PQ) and vinyl ether are functionalized into ZIKV virus and quantum dots, respectively. The ZIKV virus was then labeled as fluorescent quantum dots by cyclization under irradiation. The detailed process is described as follows:
modification of Zika virus: 9, 10-Phenanthroquinone (PQ) 11mg (4.17X 10) -5 mol) in 10ml MES buffer, 80mg EDC (4.17X)10 -4 mol) and 120mg NHS, sonicated for 15s, sealed, shaken at 37 ℃ for 15min. Then, 100. Mu.L of Zika virus (concentration: 2 mg/ml) was dispersed in 10ml of PBS. MES buffer was added, shaken at 37 ℃ overnight, and purified by NAP-5 desalting column to obtain the Zuka virus modified with PQ group. Functionalization of PQ into ZIKV viruses was achieved.
Modification of quantum dots QDs: taking the molar concentration as 8 multiplied by 10 -6 mu.L of amino group-containing quantum dots of mu.M; then, 1000. Mu.L of a vinyl ether containing carboxyl groups, 15.3mg of EDCI, and 9.8mg of 4-dimethylaminopyridine were taken out and placed in a 3-necked flask, and 5ml of dichloromethane was added thereto to be completely dissolved. Then shaken in a dark environment at room temperature for 2 hours. And then carrying out silica gel column chromatography to obtain the quantum dot modified by the vinyl ether group. The functionalization of the vinyl ether into the quantum dots is achieved.
Click reaction of Zika virus with quantum dots: dissolving PQ-modified Zika virus and vinyl ether-modified quantum dots in CH 3 And (3) irradiating the mixed solution of CN and PBS for 1min by using an LED lamp to perform cyclization reaction to mark the ZIKV virus as fluorescent quantum dots, thereby obtaining the quantum dot modified ZiKV-Qds. As shown in fig. 2 to 3, the primary absorption peak of the prepared 9, 10-phenanthrenequinone is significantly blue-shifted, the absorption band at 330nm to 310nm and 425nm disappears with the irradiation of the LED lamp, and the fluorescence intensity of the reaction of PQ with electron-rich vinyl ether at 450nm is greatly increased with the increase of the irradiation time due to the light click reaction between PQ and the electron-rich vinyl ether. Meanwhile, as shown in fig. 3, their mixed solution changed from yellow to colorless and appeared blue light, which is consistent with the aforementioned absorbance and fluorescence results. In addition, whether the quantum dots are marked on the ZIKV virus is verified by adopting a polyacrylamide gel electrophoresis method. As shown in fig. 4, ZIKV-quantum dots remain in place compared to quantum dots, which are contributed by the macromolecular proteins of the virus. On the other hand, as shown in fig. 5, the transmission electron microscope observes that the ZIKV-quantum dots have a typical quantum dot functionalized ZIKV pattern, and intuitively verifies the success of quantum dot modification in ZIKV viruses. In summary, the light-point cyclization reaction between PQ and vinyl ether can be applied to quantum dot labeling of ZIKV virus.
ZIKV-Qds internalization analysis:
arruda et al reported that the process to obtain ZIKV was optimized by a discontinuous sucrose gradient purification. To determine whether ZIKV particles with infectivity were present along the gradient fractions, all fractions were titrated by plaque. In fact, despite the presence of ZIKV in all fractions, the purified titers were above 10, as shown in fig. 6 8 pfu/ml; as shown in FIG. 7, the peak of ZIKV RNA level reached 5.0pfu/ml (specific value). Times.10 10 RNA copies/ml, peaking at a sucrose content of 20%. Quantum dots and ZIKV-quantum dots were characterized by using reflected light generated by brownian motion of nanoparticles suspended in solution in combination with NanoSight NS300 instrument and malvern instrument software. For each measurement, the concentration value (particles/ml) and the size (nm) of Zika virus, quantum dot-modified Zika virus were determined. As shown in FIG. 8, the NTA concentration of ZIKV was 2.92X 10 5 ±3.55×10 4 The average particle diameter of the particles per mL is 119.3 +/-11.5 nm. As shown in FIG. 9, the mean particle size of Zika virus was 108.9. + -. 8.2nm, which is larger than that of quantum dot-modified Zika virus (2.14X 10) 6 ±4.10×10 5 pieces/mL) was 13.6 times higher. As shown in FIG. 10, zeta potential values of the quantum dot and the ZIKV virus were-8.14 mV and-12.03 mV, respectively. Zeta potential peaks of the ZIKV-quantum dot sample are obviously separated, and the Zeta potential peaks are combined with negative charges, so that the quantum dots are successfully modified on the ZIKV surface through an ammonia bond between amino and carboxyl. As shown in fig. 11, there was no difference in the ZIKV and ZIKV-quantum dot virus titers, indicating that the quantum dots had no effect on the penetration performance of the ZIKV virus. These tests strongly suggest that quantum dots are capable of labeling ZIKV viruses without impact.
Mixing ZIKV-Qds and nucleic acid dye Syto13 in an incubator at 37 ℃ and incubating for 1h, ultracentrifuging for 2h at 108000g, removing residual dye, and purifying the purified substance for later use, or multiplying for later use according to the situation.
With QDs modified ZIKV virus, the invention further evaluates the infiltration capacity of ZIKV by fluorescence visualization of QDs. In the absence of direct evidence of ZIKV infection in cells, we used QDs-modified ZIKV to map the infection process.
An A549 cell group for incubating ZIKV and an A549 cell group for incubating ZIKV-QDs are arranged. The nucleic acid dye Syto13 was added to the group of a549 cells incubated with ZIKV, and the purified substance was added to the group of a549 cells incubated with ZIKV-QDs, shaken 5 times every 15 minutes, and then incubated for 24 hours in an incubator containing 5% carbon dioxide at 37 ℃. After completion of incubation, cells were fixed with 4% paraformaldehyde, and then fluorescence imaging was observed under a laser confocal scanning microscope (LSM 880), and the above fluorescent dye particles were photographed. The excitation wavelength of the quantum dots is 561nm, and the emission wavelength is 605nm; the excitation wavelength of Syto13 was 488nm and the emission wavelength was 509nm. As shown in fig. 12, fluorescence imaging of a549 cells incubated with ZIKV-QDs in the confocal microscope blue channel was brighter than that of a549 cells incubated with ZIKV, with no significant difference in green emission of the Syto13 dye under the two conditions. The fluorescence images of QDs overlap well with the fluorescence of the commercial nucleic acid tracking dye Syto 13. As shown in FIGS. 13-14, the Pearson correlation coefficient (PC) for the A549 cell group incubated with ZIKV-QDs was 0.95.
In order to verify the wide application of the ZIKV-QDs targeting tracing performance, the invention also provides an SNB 19 cancer cell group for incubating ZIKV and an SNB 19 cancer cell group for incubating ZIKV-QDs. The nucleic acid dye Syto13 was added to the group of SNB 19 cancer cells incubated with ZIKV, and the purified material was added to the group of SNB 19 cancer cells incubated with ZIKV-QDs, in the same manner as in the a549 cell experiment above. A remarkable fluorescence co-localization experiment was performed on the SNB 19 cancer cell line by confocal fluorescence microscopy, and the result is shown in fig. 15. Fluorescence imaging of SNB 19 cancer cells incubated with ZIKV-QDs in the confocal microscope blue channel was brighter than that of the ZIKV-incubated SNB 19 cancer cells, with no significant difference in green emission of the Syto13 dye under the two conditions. The fluorescence images of QDs overlap well with the fluorescence of the commercial nucleic acid tracking dye Syto 13. As shown in fig. 16-17, PC was also calculated to 0.95 for the SNB 19 cancer cell group that incubated ZIKV-QDs. The biological imaging results fully show that the ZIKV marked by the QDs has good positioning and tracking functions in the virus infection process.
Chlorpromazine hydrochloride (CPZ) and noridazol (norconazole) specifically inhibit clathrin-dependent endocytosis and microtubule formation, respectively, thereby preventing ZIKV infection. In view of the fact that the PQ modified quantum dots have good positioning capacity, in order to show popularization and application of the PQ modified quantum dots in therapy evaluation after ZIKV related therapy, the invention further introduces two typical antiviral drugs of chlorpromazine hydrochloride (CPZ) and noridazol. Setting a control group, a noridazol group and a CPZ group, adding the purified substances into each experimental group, wherein the noridazol group is also added with noridazol, and the CPZ group is also added with CPZ. The final results are shown in fig. 18, where the cellular fluorescence intensity of the CPZ-treated quantum dots was reduced compared to the control group due to the reduced ZIKV across the barrier cell monolayer. In addition, noridazol treatment also significantly reduced ZIKV invasion and infection 2 hours after inoculation, and noridazol significantly inhibited ZIKV entry and infection, which provided direct evidence that microtubule polymerization is essential for ZIKV intracellular transport. In view of these results, we can follow the ZIKV infection process using fluorescent nanoprobe quantum dots and evaluate the treatment effect using independent fluorescent signals.
Next, the present invention demonstrates the level of nascent protein synthesis during ZIKV infection following CPZ or noridazol (norconazole) treatment. ZIKV envelope protein E (ZIKV E) is the major structural protein exposed on the surface of granulosa cells and is thought to be involved in attachment, penetration and membrane fusion of viruses. Therefore, the reason why the ZIKV infection is inhibited by chlorpromazine hydrochloride and noconazole is researched and evaluated by selecting ZIKV E as a research object. Results of Western blotts experiments with CPZ treatment, as shown in fig. 19, indicate that ZIKV infection has a significant effect on ZIKV E synthesis, similar to that observed in cells infected with the translation inhibitor noridazol. Meanwhile, CPZ and noridazole also show some side effects as shown in fig. 20 and 21, which means that both drugs can not only inhibit microtubule polymerization but also induce apoptosis by interfering with intracellular transport. In addition, in order to test the bioapplication ability of the probe quantum dots, the cytotoxicity of the probe quantum dots was tested with cells. As shown in FIGS. 22 and 23, after incubation with quantum dots of different concentrations, no significant change in cells was observed, which indicates that the probe quantum dots can be used in living systems, and the irradiation time is different, and the effect on cells is shown in FIG. 24. In conclusion, the above results show that the ZIKV-quantum dots can accurately locate and position the ZIKV viruses by monitoring the fluorescence of the quantum dots under different conditions.
The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, the specific implementation manners and the application ranges may be changed, and in conclusion, the content of the present specification should not be construed as limiting the invention.
Claims (3)
1. A Zika virus visual marking strategy utilizing a light click bioorthogonal reaction is characterized in that:
the method comprises the following steps:
first functionalizing 9, 10-phenanthrenequinone into Zika virus, comprising: 119, 10-phenanthrenequinone mg, dispersed in 10ml MES buffer; adding 80mg EDC and 120mg NHS, performing ultrasonic action, sealing, and oscillating; then, 100. Mu.L of Zika virus at a concentration of 2mg/ml was dispersed in 10ml of PBS, and the MES buffer was added thereto and shaken;
functionalizing a vinyl ether into a quantum dot, the operations comprising: taking the molar concentration as 8 multiplied by 10 -6 Mu M quantum dot containing amino 1 mu L, then taking vinyl ether containing carboxyl 1000 mu L, EDCI 15.3mg, 4-dimethylamino pyridine 9.8mg, adding 5ml dichloromethane for complete dissolution; shaking in dark environment at room temperature;
subsequently, labeling zika virus as fluorescent quantum dots by cyclization reaction under irradiation, comprising: dissolving 9, 10-phenanthrenequinone-modified Zika virus and vinyl ether-modified quantum dots in CH 3 And (3) irradiating the mixed solution of CN and PBS for 1min by using an LED lamp to perform cyclization reaction to mark the Zika virus as fluorescent quantum dots, thereby obtaining the quantum dot modified Zika virus ZIKV-Qds.
2. The visual labeling strategy for Zika viruses by utilizing the optical click bioorthogonal reaction of claim 1, which is characterized in that:
the detailed procedures for functionalizing 9, 10-phenanthrenequinone into Zika virus include: 11 Dispersing 9, 10-phenanthrenequinone in 10ml MES buffer solution; adding 80mg EDC and 120mg NHS, performing ultrasonic action for 15s, sealing, and oscillating at 37 deg.C for 15min; then, 100. Mu.L of Zika virus at a concentration of 2mg/ml was dispersed in 10ml of PBS, and the above MES buffer was added thereto, shaken overnight at 37 ℃ and purified by a NAP-5 desalting column to obtain 9, 10-phenanthrenequinone-modified Zika virus.
3. The visual labeling strategy for Zika viruses by utilizing the optical click bioorthogonal reaction of claim 1, which is characterized in that:
the operation of functionalizing the vinyl ether into the quantum dots comprises: taking the molar concentration as 8 multiplied by 10 -6 Mu M of amino-containing quantum dots is 1 mu L, 1000 mu L of carboxyl-containing vinyl ether, 15.3mg of EDCI and 9.8mg of 4-dimethylamino pyridine are respectively put into a three-necked flask, and 5ml of dichloromethane is added to be completely dissolved; then shaking for 2 hours in a dark environment at room temperature; and then carrying out silica gel column chromatography to obtain the quantum dot modified by the vinyl ether group.
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