AIE material for labeling rabies virus as well as preparation method and application of AIE material

Abstract

The invention relates to an AIE material for marking rabies virus as well as a preparation method and application of the AIE material, and belongs to the technical field of intracellular visualization after virus infection and virus infection mechanism research. In order to obtain an AIE material capable of being used for marking rabies virus and further realize real-time visual tracking after virus infection, an alkynyl group is artificially introduced on the AIE material TPE-BP-cho through a chemical synthesis method, so that the AIE material has the capability of carrying out click reaction with amino groups. The RABV can be simply and quickly marked by using the modified AIE material, and the marked RABV not only has excellent properties of the AIE material, but also has good infection ability; and in combination with a confocal microscope, real-time tracking can be carried out in living cells after virus infection is marked, RABV intracellular transport activity can be analyzed through statistical algorithm analysis, and a good tool is provided for virus pathogenic mechanism research and antiviral drug screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of intracellular visualization after virus infection and research on virus infection mechanism, and specifically relates to an AIE material for marking rabies virus and a preparation method and application thereof, and in particular relates to an AIE material for marking rabies virus and a preparation method thereof, a method for using the AIE material to mark rabies virus and the application of the obtained rabies virus in research on virus infection mechanism and screening of antiviral drugs. Background Art

[0002] Rabies is a highly neurotropic zoonotic infectious disease caused by the rabies virus (RABV), which poses a major threat to global public health. Rabies virus can infect most warm-blooded animals. After being scratched or bitten by an infected person, RABV particles can infiltrate muscle cells through nicotinic acetylcholine receptors (nAchR) to cause primary infection. Subsequently, they cross the synaptic cleft of the neuromuscular junction (NMJ) in the peripheral nervous system. The virus undergoes retrograde axonal transport, replication and assembly in the neuronal cell body, and finally migrates to another synapse and sprouts from another synapse. This process triggers a new round of infection until the central nervous system is widely infected. Viral replication in the central nervous system can cause severe neurological diseases. So far, rabies remains an incurable disease. Once clinical symptoms appear, the mortality rate is as high as 100%. Despite centuries of research, the mechanisms of RABV infection, transmission, immune escape, and ultimately neurological diseases and death have not been thoroughly resolved. Therefore, there is an urgent need to develop a tool that can be used to study viral pathogenic mechanisms and drug screening.

[0003] RABV lacks an independent replication system and depends on entry into host cells for replication and survival. During infection, the viral life cycle begins with attachment, entry, and trafficking, during which the virus transports its genome from the cytosol entry site to the genetic replication site within the host cell, facilitating subsequent viral replication, assembly, and budding. RABV has been reported to be internalized into cells via classical dynamin-dependent clathrin-mediated endocytosis, which plays a crucial role in the viral entry process. However, the research methods for RABV infection are mainly based on electron microscopy or molecular biology, which can only obtain static images and often ignore key dynamic details during viral infection.

[0004] With the development and progress of science and technology, virus labeling technology based on fluorescence imaging provides a method to reveal virus-host dynamics. Scientists use traditional dyes, inorganic nanoparticles to label RABV or construct fluorescent proteins as fusion tags to insert into RABV to study its infection process. However, traditional organic dyes have disadvantages such as low permeability, high background autofluorescence and fluorescence quenching, and photobleaching caused by aggregation-induced fluorescence quenching effect (ACQ). The application of inorganic nanoparticles is mainly limited by their high toxicity and inappropriate size, and the process of constructing fluorescent proteins as fusion tags to insert into pathogenic microorganisms is extremely cumbersome and challenging, and may even destroy the pathogenic biological functions of pathogenic microorganisms. The discovery of aggregation-induced emission (AIE), which is the opposite of ACQ phenomenon, has greatly made up for the shortcomings of traditional biological probes. AIE materials have excellent properties such as excellent photostability, high fluorescence quantum yield and high molar absorptivity, and have received extensive attention and application in the fields of biosensors, bioimaging and bioprobes. Proteins, peptides, nucleic acids, organelles, cell surface molecules, etc. can all be labeled with AIE materials. However, when selecting AIE materials to label the labeled substances, it is necessary to select appropriate AIE materials according to the different labeled substances. For example, when selecting AIE materials to label viruses, it is necessary to meet specificity and biocompatibility, ensure that the AIE materials only bind to the target virus, and the AIE materials have minimal impact on the target virus and host cells, while ensuring good imaging effects. Therefore, the AIE materials need to be modified to meet the requirements of simple and fast labeling without affecting the luminescent properties of the AIE materials and the growth characteristics of the virus. There is currently no report on the use of AIE materials to label viruses to achieve real-time visual tracking after virus infection. Summary of the invention

[0005] In order to obtain an AIE material that can be used to label viral glycoproteins, and use it for the labeling of rabies virus, and then realize real-time visual tracking after virus infection, the present invention synthesizes a series of AIE molecules with activated alkyne function by chemical synthesis. Due to the presence of its activated alkyne group, the virus can react efficiently with it under mild conditions without a catalyst. By designing activated alkyne AIE molecules with different emission wavelengths, it can meet the labeling of rabies virus of different strains, and can also realize the labeling of different viruses. The present invention uses one of the AIE molecules as a case for labeling verification. The results show that TPE-BP-pyo simply and quickly labels RABV, and the virulence of RABV after labeling is not affected at all. At the same time, it has high fluorescence properties and strong photobleaching ability. By combining confocal microscopy (CLSM), the labeled virus can be tracked in real time in living cells after infection, and is used as a good tool for studying viral pathogenic mechanisms and screening antiviral drugs.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention specifically provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for preparing an AIE material for labeling rabies virus. Taking TPE-BP-pyo as an example, the preparation method comprises the following steps: dissolving TPE-BP-cho (570 mg, 1 mmol) in 20 mL of anhydrous tetrahydrofuran; 2 A 0.5 M solution of ethylmagnesium bromide containing THF (1.2 mmol THF dissolved in 2.4 mL ethylmagnesium bromide) was added under an atmosphere and stirred at room temperature overnight; the mixture was quenched with a saturated ammonium chloride solution, and the crude product was extracted with dichloromethane (DCM) and then precipitated with MgSO 4 The organic layer was concentrated and purified by silica gel chromatography using a mixed solution of n-hexane and DCM in a volume ratio of 2:1 to obtain a solid yellow powder; the solid yellow powder (119.2 mg, 0.2 mmol) was mixed with MnO 2 (869 mg, 10 mmol) was added to 20 mL of anhydrous DCM and heated under N 2 The mixture was stirred at room temperature for 24 h under an atmosphere of 400 μL and the compound TPE-BP-pyo was obtained by chromatography on silica gel using n-hexane / DCM, which is the AIE material.

[0008] The second object of the present invention is to provide an AIE material prepared by the above preparation method.

[0009] The third object of the present invention is to provide a method for labeling rabies virus using the above-mentioned AIE material, the method comprising the following steps:

[0010] S1. Mix TPE-BP-pyo and dimethyl sulfoxide in a ratio of 1 g: 100 mL to obtain a solution containing AIE material;

[0011] S2. Mix the purified rabies virus with the solution containing the AIE material obtained in S1 at a volume ratio of 10:1 and combine at room temperature for 1-2 hours.

[0012] The fourth object of the present invention is to provide rabies virus labeled with AIE material obtained by the above method.

[0013] The fifth object of the present invention is to provide the use of the above-mentioned AIE material or the above-mentioned labeling method or the rabies virus labeled with the above-mentioned AIE material in realizing real-time tracking of rabies virus in living cells.

[0014] In one embodiment of the present invention, RABV labeled with AIE materials was used to infect NA cells with MOI=1, and the fluorescence signal was monitored by laser scanning confocal microscopy at 1h, 2h and 3h after infection. It was found that as time passed, the virus particles continued to enter the cells, and most of the viruses completely entered the cells 3h after infection. The labeling method provided by the present invention is different from the traditional tracing method, and RABV particles can be tracked in real time without the use of antibodies. After proving that real-time imaging can be achieved by labeling RABV with the AIE material provided by the present invention, we infected NA cells and BSR cells with MOI=5 to achieve real-time visual tracking of single virus particles. It is worth mentioning that the present invention directly confirmed that the transport of RABV in axons is carried out in both directions at the same time. In general, the method of labeling RABV with AIE materials provided by the present invention can dynamically track the virus in real time in living cells, and RABV labeled with AIE materials can be used as a powerful tool for tracing single RABV particles, which helps us study the intracellular transport process and mechanism after RABV infection.

[0015] The sixth object of the present invention is to provide the use of the above-mentioned AIE material or the above-mentioned labeling method or the rabies virus labeled with the above-mentioned AIE material in studying the infection mechanism of rabies virus.

[0016] In one embodiment of the present invention, the position of the fluorescent spot in the time-lapse image is tracked by single particle tracking technology, thereby obtaining the two-dimensional trajectory of a single virus particle in the cell. Data were collected from more than 30 cells of different cell lines (NA cells and BSR cells), and at least 300 virus particle trajectories were analyzed. The movement duration of these trajectories exceeded 100 seconds, and the time resolution was 10 seconds. Based on the trajectories obtained by the present invention, we conducted a detailed study on the intracellular diffusion dynamics of RABV. We found that RABV infection has similar diffusion behavior in different cells, but there are differences in diffusion speed. SRV9 showed similar diffusion speeds in NA cells or BSR cells, while CVS-11 had a significantly higher diffusion speed in NA cells than in BSR cells. Further studies have shown that the diffusion speed, growth characteristics and early intracellular viral RNA levels of RABV are positively correlated. These findings indicate that the intracellular diffusion speed of RABV may be one of the reasons why different RABV strains have significant differences in cell tropism and pathogenesis. These research results successfully demonstrate the application prospects of the present invention in the study of viral infection mechanisms.

[0017] The seventh object of the present invention is to provide the use of the above-mentioned AIE material or the above-mentioned labeling method or the rabies virus labeled with the above-mentioned AIE material in the screening of anti-rabies virus drugs.

[0018] In one embodiment of the present invention, the present invention uses AIE material-labeled RABV to infect NA cells treated with antiviral drug-GSK126 at MOI=5, and observes the transport of RABV in NA cells in real time, revealing the effect of GSK126 on RABV infection dynamics. We further analyzed the diffusion behavior of RABV in NA cells treated with GSK126. Examination of CVS-11 trajectories in individual cells showed that compared with untreated cells, the proportion of almost motionless trajectories in GSK126-treated cells was higher, long displacements were less, and the decay of rest time distribution was slower, indicating that CVS-11 showed a greater tendency to be immobile in GSK126-treated cells, which led to an overall decrease in the diffusion rate of CVS-11 in NA cells. By fitting the EA-MSD, we found that the diffusion coefficient of CVS-11 in NA cells treated with GSK126 was only half of that in NA cells not treated with GSK126. These results demonstrate that the present invention has great potential in antiviral drug screening, and is expected to accelerate the drug screening process and promote the study of antiviral mechanisms.

[0019] In one embodiment of the present invention, the above-mentioned application of studying the rabies virus infection mechanism and anti-rabies virus drug screening includes inoculating cells with rabies virus labeled with AIE materials, and performing statistical analysis on the motion trajectory, time-averaged mean square displacement, ensemble-averaged mean square displacement, and displacement probability density distribution of all virus particles in a single cell.

[0020] Further definition, the mean square displacement statistical method is as follows: We use <Δr(t)2=<r(t)2-r(0)2> The ensemble average mean square displacement (EA-MSD) <Δr(τ)2> is calculated, where r is the two-dimensional (2D) position, t is the lag time, and <·> represents the average of many starting states (t=0). For two-dimensional diffusion, the relationship between <Δr(t)2> and t can be fitted as <Δr(t)2>=4Dtξ, where ξ is an exponent and D is the diffusion coefficient. The value of ξ is one of the indicators to determine whether the diffusion is Fickan diffusion or anomalous. ξ>1 indicates superdiffusion, ξ=1 indicates Fickan diffusion, and ξ<1 indicates subdiffusion. The TA-MSD of the virus particle is calculated as follows, where T is the trajectory length and Δ is the lag time.

[0021]

[0022] Further defined, the statistical method of the probability distribution of the ensemble mean displacement is as follows:

[0023] Another important quantity to characterize diffusion is the ensemble mean displacement probability distribution (DPD, P(x, t)), which represents the probability that an object moves a distance x in one dimension during time t. We calculate it based on the self-part of the van Hove correlation function.

[0024]

[0025] Where N is the number of trajectories and the angle brackets <·> denote the trajectory average.

[0026] In one embodiment of the present invention, the RABV may also be any enveloped virus. Those skilled in the art should understand that the AIE material labeling principle is to label the RABV G protein. This labeling method is non-specific, so in theory any enveloped virus with a G protein can be labeled, including but not limited to RABV.

[0027] In some specific embodiments of the present invention, the RABV is a CVS-11 strain and an SRV-9 strain. However, those skilled in the art will appreciate that the CVS-11 strain represents a standard attack strain and the SRV-9 strain represents a standard vaccine strain, but the RABV of the present invention is not limited to the CVS-11 strain and the SRV-9 strain, and may be any strain (e.g., Flury strain, PV strain, PM strain, etc.).

[0028] In some specific embodiments of the present invention, the NA cells and BSR cells, those skilled in the art should understand that NA cells are neuroblastoma cells, which can represent neural cells, and BSR cells are human milk hamster kidney passage cells, which can represent somatic cells.

[0029] In some specific embodiments of the present invention, the AIE material is TPE-BP-pyo. However, those skilled in the art should understand that since AIE materials have a high degree of similarity, the AIE material of the present invention is not limited to TPE-BP-pyo, and can be any AIE material having an activated alkyne group.

[0030] Beneficial effects of the present invention:

[0031] The present invention simply and quickly labels RABV by using an AIE material introduced with an alkynyl group, and obtains RABV with both good infection ability and good optical properties of AIE materials, successfully avoiding the problems of low permeability, high background autofluorescence and fluorescence quenching, photobleaching caused by the aggregation of traditional organic dyes, high toxicity and inappropriate size of inorganic nanoparticles, and the complicated operation of constructing fluorescent proteins as fusion tags to insert pathogenic microorganisms, and even destroying pathogenic biological functions. The present invention uses RABV labeled with AIE materials, and by combining confocal microscopy (CLSM), the movement trajectory of RABV is tracked in real time in living cells, and the obtained trajectory is analyzed, and the intracellular dynamics of viral infection are studied in detail, which promotes the understanding of the intracellular transport mechanism of RABV, and provides new tools and ideas for revealing the complexity of viral pathogenesis. At the same time, the RABV labeled with AIE materials provided by the present invention has great application potential in antiviral drug screening and is expected to accelerate the drug screening process. In summary, the RABV labeled with the AIE material provided by the present invention can be used as a good tool for studying viral pathogenic mechanisms and screening antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the molecular structure formula of TPE-BP-pyo;

[0033] Figure 2 This is the result of the capability verification of AIE materials labeling BSA protein and rabies virus strain CVS-11, where: Figure 2 A in the figure is the UV irradiation result of gel imaging of BSA protein labeled with AIE material. Figure 2 Figure B is the Coomassie brilliant blue staining result of BSA protein labeled with AIE material. Figure 2 C in the figure is the UV irradiation result of gel imaging of CVS-11 after AIE material labeling and purification. Figure 2 D in the figure is the Coomassie brilliant blue staining result of CVS-11 after AIE material labeling and purification; Figure 2 In A and B, M is a marker, 1 is 500 μg BSA, 2 is 50 μg AIEgen + 500 μg BSA, 3 is 50 μg AIEgen, Figure 2 In C and D, M is Marker, 1 is 10 μg AIEgen, 2 is 100 μg CVS-11, and 3 is 10 μg AIEgen + 100 μg CVS-11;

[0034] Figure 3 The figure is a test result diagram of the titer of unlabeled rabies virus and rabies virus labeled with AIE material;

[0035] Figure 4 The fluorescence property test results of rabies virus, AIE material and rabies virus labeled with AIE material are shown in FIG. Figure 4 A in FIG. 1 is the absorption spectra of rabies virus, AIE material, and rabies virus labeled with AIE material. Figure 4 B in the figure is the emission spectra of rabies virus, AIE material and rabies virus labeled with AIE material;

[0036] Figure 5 The figure is a verification result diagram of RABV labeled with AIE material by autofluorescence; Figure 5 A in the figure is a picture of Confocal verifying the labeled virus particles in vitro, the red light is AIEgen, and the green light is RABV G; Figure 5 B in the figure is the Confocal verification of the labeled virus particles, the red light is AIEgen, and the green light is Dio dye; Figure 5 C in the figure is a picture of Confocal verifying the labeled virus particles in NA cells, the red light is AIEgen, and the green light is RABV G;

[0037] Figure 6 This is a diagram showing the application results of AIE-labeled RABV for real-time tracking of RABV in cells; Figure 6 A in the figure is the result of Confocal verifying whether the AIEgen-labeled virus can be visualized in cells without the need for antibodies 1h, 2h, and 3h after infection. Figure 6 Figure B is the application result of single particle tracking of CVS-11 strain in NA cells. Figure 6 Figure C is the result of single particle tracking of CVS-11 strain in BSR cells. Figure 6 D in the figure is the application result of single particle tracking of SRV-9 strain in NA cells. Figure 6 Figure E is the application result of single particle tracking of SRV-9 strain in BSR cells. Figure 6 F in the figure is a tracking diagram of the bidirectional transport trajectory of virus particles in the axons of NA cells;

[0038] Figure 7 This is a diagram showing the movement analysis of all CVS-11 virus particles in a single NA cell, where: Figure 7 A in the figure is the movement trajectory of all CVS-11 virus particles in a single NA cell. Figure 7 B in the figure is a statistical graph of the time-averaged mean square displacement of all CVS-11 virus particles in a single NA cell. Figure 7 C in the figure is the statistical graph of the ensemble average mean square displacement of all CVS-11 virus particles in a single NA cell. Figure 7D in the figure is the displacement probability density distribution statistical diagram of all CVS-11 virus particles in a single NA cell;

[0039] Figure 8 This is a diagram showing the movement analysis of all CVS-11 virus particles in a single BSR cell, where: Figure 8 A in the figure is the movement trajectory of all CVS-11 virus particles in a single BSR cell. Figure 8 B in the figure is the statistical graph of the time-averaged mean square displacement of all CVS-11 virus particles in a single BSR cell. Figure 8 C in the figure is the statistical graph of the ensemble average mean square displacement of all CVS-11 virus particles in a single BSR cell. Figure 8 D in the figure is the displacement probability density distribution statistical diagram of all CVS-11 virus particles in a single BSR cell;

[0040] Fig. 9 is the ensemble average MSD graph and displacement probability density distribution graph of CVS-11 in different cell lines, where: Fig. 9 The red line in A in Figure 1 represents the ensemble average mean square displacement of all CVS-11 particles in each NA cell, and the black line represents the ensemble average mean square displacement of all CVS-11 particles in all statistical NA cells. Fig. 9 The red line in B represents the ensemble average mean square displacement of all CVS-11 particles in each BSR cell, and the black line represents the ensemble average mean square displacement of all CVS-11 particles in all statistical BSR cells. Fig. 9 In Figure C, the black circles represent the displacement probability density distribution statistics of all CVS-11 virus particles in BSR cells, and the red circles represent the displacement probability density distribution statistics of all CVS-11 virus particles in NA cells; Fig. 9 In D, the red line represents the ensemble average mean square displacement of all CVS-11 particles in all statistical NA cells, and the black line represents the ensemble average mean square displacement of all CVS-11 particles in statistical BSR cells;

[0041] Fig.10 This is a diagram showing the movement analysis of all SRV-9 virus particles in a single NA cell, where: Fig.10 A in the figure is the movement trajectory of all SRV-9 virus particles in a single NA cell. Fig.10 B in the figure is a statistical graph of the time-averaged mean square displacement of all SRV-9 virus particles in a single NA cell. Fig.10 C in the figure is the statistical graph of the ensemble average mean square displacement of all SRV-9 virus particles in a single NA cell. Fig.10 D in the figure is the distribution statistical diagram of the displacement probability density of all SRV-9 virus particles in a single NA cell;

[0042] Fig.11 This is a diagram showing the movement analysis of all SRV-9 virus particles in a single BSR cell, where: Fig.11 A in the figure is the movement trajectory of all SRV-9 virus particles in a single BSR cell. Fig.11 B in the figure is the statistical diagram of the time-averaged mean square displacement of all SRV-9 virus particles in a single BSR cell. Fig.11 C in the figure is the statistical graph of the ensemble average mean square displacement of all SRV-9 virus particles in a single BSR cell. Fig.11 D in the figure is the displacement probability density distribution statistical diagram of all SRV-9 virus particles in a single BSR cell;

[0043] Fig.12 is the ensemble average MSD graph and displacement probability density distribution graph of SRV-9 in different cell lines, where: Fig.12 The red line in A in Figure 1 represents the ensemble average mean square displacement of all SRV-9 particles in each NA cell, and the black line represents the ensemble average mean square displacement of all SRV-9 particles in all statistical NA cells. Fig.12 The red line in B represents the ensemble average mean square displacement of all SRV-9 particles in each BSR cell, and the black line represents the ensemble average mean square displacement of all SRV-9 particles in all statistical BSR cells. Fig.12 In Figure C, the black circles represent the displacement probability density distribution statistics of all SRV-9 virus particles in BSR cells, and the red circles represent the displacement probability density distribution statistics of all SRV-9 virus particles in NA cells; Fig.12 In D, the red line represents the ensemble average mean square displacement of all SRV-9 particles in all statistical NA cells, and the black line represents the ensemble average mean square displacement of all SRV-9 particles in statistical BSR cells;

[0044] Fig.13 The results of the RABV N mRNA level detection in NA cells not treated with GSK126 and NA cells treated with GSK126 are shown in the figure;

[0045] Fig.14 This is a statistical result of the number of CVS-11 viral ions entering NA cells that were not treated with GSK126 and NA cells that were treated with GSK126;

[0046] Fig.15 This is a diagram showing the movement analysis of all CVS-11 virus particles in a single NA cell treated with GSK126, where Fig.15 A in the figure is the movement trajectory of all CVS-11 virus particles in a single NA cell treated with GSK126. Fig.15B is a statistical graph of the time-averaged mean square displacement of all CVS-11 virus particles in a single NA cell treated with GSK126. Fig.15 C in the figure is the statistical graph of the ensemble average mean square displacement of all CVS-11 virus particles in a single NA cell treated with GSK126. Fig.15 D in the figure is the displacement probability density distribution statistical diagram of all CVS-11 virus particles in a single NA cell treated with GSK126;

[0047] Fig.16 It is the ensemble average mean square displacement statistical diagram and displacement probability density distribution diagram of CVS-11 strain in NA cells treated with GSK126 and NA cells not treated with GSK126; among them, Fig.16 A in the figure is the statistical graph of the ensemble average mean square displacement of CVS-11 strain in NA cells treated with GSK126 and NA cells not treated with GSK126. Fig.16 B in the figure is the displacement probability density distribution diagram of CVS-11 strain in NA cells treated with GSK126 and NA cells not treated with GSK126. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings.

[0049] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0050] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0051] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0052] The experimental materials used in the following examples are as follows:

[0053] 1. Cells: NA cells (mouse neuroma cells) and BSR cells (human lactating hamster kidney passage cells) are both preserved by the applicant and can also be purchased through commercial channels.

[0054] 2. Virus: Rabies virus standard attack strain CVS-11, rabies virus vaccine strain SRV-9, stored at -80℃.

[0055] 3. Culture medium: DMEM culture medium containing 10% (v / v) fetal bovine serum (FBS), penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL.

[0056] The virus culture and assay methods used in the following examples are as follows:

[0057] 1. Virus culture method:

[0058] (1) NA cells were subcultured into T25 cell culture flasks at a cell density of 2×10 5 / mL, placed at 37°C in a 5% CO 2 Cultured overnight in an incubator;

[0059] (2) The cultured cells were washed once with PBS, infected with rabies virus CVS-11 or SRV-9 at an MOI of 0.1, and incubated at 37°C for 1 h;

[0060] (3) After discarding the virus solution, wash twice with PBS, add DMEM medium containing 10% FBS, and place at 37°C with 5% CO 2 Cultured in an incubator for 72h;

[0061] (4) After 72 h, the supernatant in the culture flask was collected and centrifuged at 3000 × g for 15 min at 4°C to remove cell debris. The viral supernatant was then transferred to a 1.5 mL EP tube and stored in a -80°C refrigerator for later use.

[0062] 2. Virus culture titer determination method:

[0063] (1) Dilute the viral supernatant to be titered at a ratio of 1:10 and continue diluting until 10 9 Finally, 4 replicate wells were set for each dilution, and the virus dilutions were added to the 96-well plate containing NA cells;

[0064] (2) Place the 96-well plate in a 37°C, 5% CO 2After continuing to culture in an incubator for 48 hours, the supernatant was discarded, and the cells were fixed with 80% cold acetone at room temperature for 30 minutes, washed three times with PBST, and diluted (1:500) RABV N protein antibody with FITC fluorescence was added. After culture in an incubator at 37°C for 1 hour, the cells were washed three times with PBST, and finally observed under a fluorescence microscope and the results were recorded to calculate the virus titer.

[0065] 3. Method for drawing virus growth curve:

[0066] (1) NA cells were passaged into 6-well plates at a cell density of 2×10 5 / mL, placed at 37°C in a 5% CO 2 Cultured overnight in an incubator;

[0067] (2) The cultured cells were washed once with PBS, infected with rabies virus CVS-11 or SRV-9 at an MOI of 0.1, and incubated at 37°C for 1 h;

[0068] (3) After discarding the virus solution, wash twice with PBS, add DMEM medium containing 10% FBS, and place at 37°C with 5% CO 2 Incubator, virus supernatants were collected on days 1, 2, 3, and 4 after infection.

[0069] (4) The virus supernatant is titered according to the above method and a virus growth curve is drawn.

[0070] The SDS-PAGE method used in the following examples is as follows:

[0071] (1) Take the same amount of protein from each sample into a clean 1.5 mL centrifuge tube, add a certain amount of 5× SDS loading buffer, and boil for 7 min;

[0072] (2) Prepare 10% SDS-PAGE separation gel solution and quickly pour it into the gap between the two glass plates of the gel casting apparatus. Gently cover the separation gel with a layer of anhydrous ethanol and let it stand at room temperature for more than 30 min.

[0073] (3) Prepare 5% SDS-PAGE stacking gel solution, quickly pour it onto the separation gel, and then immediately insert a 10-hole comb and let it stand at room temperature for more than 30 minutes;

[0074] (4) After the concentrated gel is completely coagulated, place the gel caster into the electrophoresis tank, add 1× electrophoresis buffer, and carefully remove the comb;

[0075] (5) Add protein samples and protein markers in a predetermined order, add an equal volume of 1× loading buffer to the excess wells for balance, adjust the voltage to 120 V and perform electrophoresis until bromophenol blue reaches or runs out of the bottom of the gel.

[0076] The confocal observation method used in the following embodiments is as follows:

[0077] (1) NA cells were passaged into confocal microplates at a cell density of 1:6 one day in advance, with a cell density of 5×10 4 / mL, placed at 37°C in a 5% CO 2 Cultured overnight in an incubator;

[0078] (2) The cultured cells were washed once with PBS, infected with rabies virus CVS-11 or SRV-9 at an MOI of 5, and incubated at 37°C for 1 h;

[0079] (3) After discarding the virus solution, wash twice with PBS, add DMEM medium containing 10% FBS, and place at 37°C with 5% CO 2 Incubator, turn on the CO at the desired time point after infection. 2 Confocal microscope in a workroom (37°C, 5% CO 2 ) for observation and shooting.

[0080] The indirect immunofluorescence method used in the following examples is as follows:

[0081] (1) NA cells were passaged into confocal microplates at a cell density of 1:6 one day in advance, with a cell density of 5×10 4 / mL, placed at 37°C in a 5% CO 2 Cultured overnight in an incubator;

[0082] (2) The cultured cells were washed once with PBS, infected with rabies virus CVS-11 or SRV-9 at an MOI of 5, and incubated at 37°C for 1 h;

[0083] (3) After discarding the virus solution, wash twice with PBS, add DMEM medium containing 10% FBS, and place at 37°C with 5% CO 2 After observation, the confocal dish cell culture surface was removed and placed in a six-well plate;

[0084] (4) Add 700 μL of 4% paraformaldehyde, fix at room temperature for 30 min, discard the paraformaldehyde, and wash three times with PBS;

[0085] (5) Use 0.2% volume fraction of Triton for permeabilization, let it stand at room temperature for 5 min, discard the Triton, and wash with PBS three times;

[0086] (6) Then, 1% BSA was used for blocking. After standing at room temperature for 30 min, RABV G antibody (1:300) was added. After standing at 37°C for 60 min, the cells were washed three times with PBST. Finally, a secondary antibody containing FITC (1:300) was added. After standing at 37°C in the dark for 60 min, the cells were washed three times with PBST and observed under a confocal microscope.

[0087] The real-time fluorescence quantitative PCR (RT-qPCR) method used in the following examples is as follows:

[0088] (1) NA cells were passaged into 12-well plates at a cell density of 1×10 5 / mL, placed at 37°C in a 5% CO 2 Incubate overnight in an incubator;

[0089] (2) The cultured cells were washed once with PBS, infected with rabies virus CVS-11 or SRV-9 at an MOI of 0.1, and incubated at 37°C for 1 h;

[0090] (3) After discarding the virus solution, wash twice with PBS, add DMEM medium containing 10% FBS, and place at 37°C with 5% CO 2 Incubator, discard the virus supernatant 1-12 h after infection, collect the cells, add 500 μL Trizol to lyse the cells and extract total RNA;

[0091] (4) Total RNA extraction: ① Add 100 μL of chloroform to 500 μL of Trizol-lysed cells, shake and mix, and let stand at room temperature for 5 min; ② Centrifuge at 4°C and 12,000×g for 10 min, carefully aspirate 150 μL of the supernatant and add it to a clean 1.5 mL EP tube free of RNase, add an equal volume of isopropanol, and gently shake and mix; ③ Let the mixture stand on ice for 15 min; ④ Centrifuge at 4°C and 12,000×g for 10 min, and discard the filtrate; ⑤ Add 1 mL of washing solution (75% ethanol solution diluted with DEPC water) to the preparation tube, centrifuge at 4°C and 10,000×g for 5 min, and discard the filtrate; ⑥ Repeat the wash once; ⑦ Discard the filtrate and air-dry the precipitate until it is translucent; ⑧ Dissolve it in 30 μL of RNase-free water, measure the RNA concentration for reverse transcription or store at -80°C for later use;

[0092] (5) Reverse transcription: reverse transcription was performed using a reverse transcription kit under the following reaction conditions: 37°C for 15 min and 85°C for 5 s. After reverse transcription into cDNA, real-time fluorescence quantitative PCR amplification was performed or the cDNA was stored at -20°C for future use.

[0093] (6) Real-time fluorescence quantitative PCR amplification: To detect the expression of rabies virus gene mRNA, the reaction conditions were as follows: 50°C, 2 min; 95°C, 2 min; 95°C, 15 s, 58°C, 15 s, 72°C, 1 min, for a total of 40 cycles; 95°C, 15 s, 60°C, 1 min, 95°C, 15 s.

[0094] The analytical methods used in the following examples are as follows:

[0095] 1. Trajectory statistics method

[0096] To obtain the trajectory of the particles, we use program code to perform a single particle tracking algorithm on each of the numerous frames obtained from the laser scanning confocal microscope. We first enhance the signal-to-noise ratio of the original image, including contrast adjustment, sharpening, and other processing techniques. Subsequently, we process all images in a binarized and blurred manner. After this, we locate the fluorescent particles. Since the diffraction limit of light limits the resolution of the microscope, each particle is imaged as a blurred point of light, called a point spread function (PSF). By fitting the PSF with a Gaussian model, we can get the center position of the fluorescent particle and obtain the coordinates of the target particle in the image. Next, we use the nearest neighbor algorithm to identify the same particles in different frames. Finally, according to the time order, we connect the changing positions of these identified particles over time to form a time series trajectory.

[0097] 2. Mean square displacement statistical method

[0098] We use <Δr(t)2=<r(t)2-r(0)2> The ensemble average mean square displacement (EA-MSD) <Δr(τ)2> is calculated, where r is the two-dimensional (2D) position, t is the lag time, and <·> represents the average of many starting states (t=0). For two-dimensional diffusion, the relationship between <Δr(t)2> and t can be fitted as <Δr(t)2>=4Dtξ, where ξ is an exponent and D is the diffusion coefficient. The value of ξ is one of the indicators to determine whether the diffusion is Fickan diffusion or anomalous. ξ>1 indicates superdiffusion, ξ=1 indicates Fickan diffusion, and ξ<1 indicates subdiffusion. The TA-MSD of the virus particle is calculated as follows, where T is the trajectory length and Δ is the lag time.

[0099]

[0100] 3. Statistical method of probability distribution of ensemble mean displacement

[0101] Another important quantity to characterize diffusion is the ensemble mean displacement probability distribution (DPD, P(x, t)), which represents the probability that an object moves a distance x in one dimension during time t. We calculate it based on the self-part of the van Hove correlation function.

[0102]

[0103] Where N is the number of trajectories and the angle brackets <·> denote the trajectory average.

[0104] Example 1: Preparation of AIE materials for labeling rabies virus

[0105] TPE-BP-cho (570 mg, 1 mmol) purchased from the Institute for Advanced Study on Aggregation-Induced Emission was dissolved in 20 mL of anhydrous tetrahydrofuran. 2 Under the atmosphere, a 0.5 M solution of ethylmagnesium bromide containing THF (1.2 mmol THF dissolved in 2.4 mL ethylmagnesium bromide) was slowly added and stirred at room temperature overnight. The mixture was quenched with a saturated ammonium chloride solution, and the crude product was extracted with dichloromethane (DCM) and precipitated with MgSO 4 The organic layer was dried and concentrated and purified by silica gel chromatography. The compound was obtained as a solid yellow powder by gel chromatography with n-hexane / DCM (2:1, v / v). The solid yellow powder (119.2 mg, 0.2 mmol) and MnO 2 (869 mg, 10 mmol) were added to 20 mL of anhydrous DCM and stirred under N 2 The mixture was stirred at room temperature for 24 h under an atmosphere, and then the compound TPE-BP-pyo (molecular structure is shown in Figure 1 ).

[0106] Example 2: Method for labeling rabies virus using AIE material for labeling rabies virus

[0107] Pretreatment of AIEgen: A solution containing the compound TPE-BP-pyo was prepared by dissolving 10 mg of TPE-BP-pyo in 1 mL of dimethyl sulfoxide (DMSO).

[0108] 10 mL of purified CVS-11 was combined with 1 mL of AIEgen (a solution containing TPE-BP-pyo) at room temperature for 2 hours to obtain the rabies virus strain CVS-11 labeled with AIEgen.

[0109] To study the effect of AIEgen labeling, the present invention conducted the following experiments:

[0110] 1. Verification of AIEgen’s ability to label proteins and viruses

[0111] After 50 μL BSA (50 μg) protein was combined with 5 μL AIEgen (solution containing TPE-BP-pyo) at room temperature for 2 h, SDS-PAGE electrophoresis was performed, followed by UV irradiation using the Qinxiang gel imaging system and Coomassie brilliant blue staining. The results showed that AIEgen could well label BSA (see Figure 2 A and B in the figure). 10 mL of purified CVS-11 was combined with 1 mL of AIEgen (solution containing TPE-BP-pyo) at room temperature for 2 h, and then subjected to SDS-PAGE electrophoresis. The Qinxiang gel imaging system was used for UV irradiation and Coomassie brilliant blue staining. The results showed that AIEgen could also well label the purified virus (see Figure 2 C and D in ).

[0112] 2. Verification of the fluorescence properties and infectivity of viruses after AIEgen labeling

[0113] RABV was divided into two groups: CVS-11 group and CVS-11+AIEgen group, where the working concentration of AIEgen was 100 μg / mL. After flipping and binding for 1 hour at room temperature, the two experimental groups were centrifuged at 3000 rpm for 10 minutes to remove impurities such as cell debris, followed by ultracentrifugation at 30000 rpm for 1.5 hours, and then concentrated to remove unbound AIEgen.

[0114] The virus titer of the concentrated and purified CVS-11 was measured, and the results showed that the virulence of the concentrated AIEgen-labeled virus was slightly lower than that of the concentrated virus without AIEgen labeling, but there was no significant difference (see Figure 3 ), indicating that AIEgen labeling of the virus does not affect the virulence of the virus. Subsequently, the emission and absorption spectra of AIEgen, AIEgen-labeled virus, and unlabeled virus were measured, and the results showed that AIEgen labeling of the virus did not affect the absorption and emission of AIEgen (see Figure 4 ). That is, AIEgen does not affect the infectivity of the virus, and the virus does not affect the optical properties of AIEgen.

[0115] 3. Validation of autofluorescence for AIEgen-labeled RABV

[0116] RABV was divided into three groups: CVS-11 group, CVS-11+AIEgen group and CVS-11+AIEgen+Dio group. Dio is a commercialized lipophilic membrane dye, whose full name is: 3,3′-dioctadecyloxacarbocyanineperchlorate. It is one of the most commonly used cell membrane fluorescent probes and exhibits green fluorescence. The working concentration of AIEgen is 100μg / mL, and the working concentration of Dio is 10μM. After flipping and binding for 1h at room temperature, the three experimental groups were centrifuged at 3000rpm for 10min to remove impurities such as cell debris, and then ultracentrifuged at 30000rpm for 1.5h to concentrate and remove unbound AIEgen.

[0117] The purified AIEgen-labeled CVS-11 was co-incubated with RABV G protein antibody at 37°C for 1 h, and an indirect immunofluorescence experiment was performed. Finally, laser scanning confocal microscopy was used to observe the results. The results showed that AIEgen could well label most CVS-11 (see Figure 5 We also used Dio, a dye that has been reported to effectively label enveloped viruses, to stain AIEgen-labeled CVS-11. The results showed that the fluorescence of AIEgen and Dio was clearly co-localized (see Figure 5 Finally, we infected NA cells with labeled RABV that emits red light, fixed the cells and performed indirect immunofluorescence experiments. The results showed that the fluorescence of AIEgen and RABV G protein were clearly co-localized (see Figure 5 C), all results indicate that autofluorescence represents RABV.

[0118] Example 3: Application of RABV labeled with AIE materials in real-time virus tracking in living cells

[0119] In order to explore whether AIEgen-labeled RABV can be visualized in real time in living cells, the present invention first inoculated NA cells with AIEgen-labeled CVS-11 strains and AIEgen and Dio double-labeled CVS-11 strains at an inoculation amount of MOI=5, and took samples 1h, 2h and 3h after infection for direct observation by laser scanning confocal microscopy. The results showed that both single-labeled viruses and double-labeled viruses have the ability to infect, and the virus particles continue to enter the cells over time. Moreover, the virus particles can be visualized without the use of RABV-related antibodies, which provides a basis for the real-time visualization of virus particles in living cells (see Figure 6After proving that the AIE material provided by the present invention has the ability of real-time imaging, we used CVS-11 strain and SRV-9 strain to infect NA cells and BSR cells at MOI=5 to achieve real-time visual tracking of single virus particles (see Figure 6 It is worth mentioning that the present invention directly confirms that the transport of RABV in axons is bidirectional and simultaneous (see Figure 6 F).

[0120] Example 4: Application of RABV labeled with AIE materials in the study of viral infection mechanism

[0121] NA cells were inoculated with AIEgen-labeled CVS-11 strain at an MOI of 5, and the motion trajectories, time-averaged mean square displacement, ensemble-averaged mean square displacement, and displacement probability density distribution of all virus particles in a single NA cell were statistically analyzed (see Figure 7 Similarly, BSR cells were inoculated with AIEgen-labeled CVS-11 strain at MOI=5, and the movement of CVS-11 in BSR cells was observed in real time 3 h after infection. The movement trajectory, time-averaged mean square displacement, ensemble-averaged mean square displacement, and displacement probability density distribution of all virus particles in a single BSR cell were statistically analyzed (see Figure 8 By comparing the movement patterns of CVS-11 in neural cells (NA) and somatic cells (BSR), we found that there were obvious differences in their movement rates. To explore whether this difference is universal, we analyzed a large number of samples and performed statistical analysis on them. We obtained the ensemble mean square displacement of RABV CVS-11 strains in neural cells and somatic cells, and plotted the displacement probability density distribution of the two cell lines (see Fig. 9 ), we compared the ensemble average mean square displacement of CVS-11 in different cell lines, and the results showed that the diffusion coefficient of CVS-11 in neurons was 0.00248±0.00022μm 2 / s, while the diffusion coefficient in the body cell is 0.00110±0.00007μm 2 / s, and the diffusion coefficient of CVS-11 in nerve cells is twice that in body cells. The same method was used to infect NA and BSR cells with the labeled SRV9 virus strain, and the movement trajectory, time-averaged mean square displacement, ensemble-averaged mean square displacement, and displacement probability density distribution of all virus particles in a single NA cell were statistically analyzed (see Fig.10Similarly, BSR cells were inoculated with AIEgen-labeled SRV9 strain at MOI=5, and the movement of SRV9 in BSR cells was observed in real time 3 h after infection. The movement trajectory, time-averaged mean square displacement, ensemble-averaged mean square displacement, and displacement probability density distribution of all virus particles in a single BSR cell were statistically analyzed (see Fig.11 By comparing the movement patterns of SRV9 in neural cells (NA) and somatic cells (BSR), we found that unlike the movement of CVS-11 in different cell lines, the movement rate of SRV9 in different cell lines was not significantly different. In order to explore whether this phenomenon is universal, we also analyzed a large number of samples and performed statistical analysis on them, and obtained the ensemble average mean square displacement of RABV SRV9 strains in neural cells and somatic cells, and at the same time drew the displacement probability density distribution diagram of the two cell lines (see Fig.12 ), we compared the ensemble average mean square displacement of SRV9 in different cell lines, and the results showed that the diffusion coefficient of SRV9 in neurons was 0.00228±0.00015μm 2 / s, and the diffusion coefficient in the body cell is 0.00241±0.00009μm 2 / s, the diffusion coefficient of SRV9 is not much different whether in nerve cells or somatic cells. These results successfully demonstrate the application of AIE material labeled RABV in the study of RABV infection mechanism.

[0122] Example 5: Application of RABV labeled with AIE materials in antiviral drug screening

[0123] NA cells were treated with an inhibitor (GSK126) at a working concentration of 2 μM for 12 h, and then inoculated with RABV CVS-11 strain at an MOI of 0.1. RNA was extracted 24 h after infection, and the changes in RABV N mRNA in the treatment group and the control group were detected by qPCR. The results showed that the RABV N mRNA level in the inhibitor treatment group at a working concentration of 2 μM was significantly decreased compared with the control group (see Fig.13 ). Then, NA cells treated with the inhibitor at a working concentration of 2 μM for 12 h were inoculated with AIEgen-labeled CVS-11 strain at an MOI of 5. The status of CVS-11 in NA cells was observed in real time 3 h after infection. We found that the number of virus particles entering NA cells treated with GSK126 was reduced (see Fig.14 ), the trajectory of all virus particles in a single NA cell (see Fig.15 A in), time-averaged mean square displacement (see Fig.15 B in), ensemble mean square displacement ( Fig.15 C) and the displacement probability density distribution (see Fig.15By comparing the movement patterns of CVS-11 in untreated neurons and neurons treated with inhibitors, we found that the movement rate of CVS-11 in neurons treated with inhibitors was significantly reduced. In order to explore whether this phenomenon is universal, we analyzed a large number of samples and conducted statistical analysis on them, and obtained the ensemble average mean square displacement ( Fig.16 A in the figure, and plotted its displacement probability density distribution diagram ( Fig.16 In Figure B), we compared the ensemble average mean square displacement of CVS-11 in neurons treated with and without inhibitors. The results showed that the diffusion coefficient of CVS-11 in neurons treated with inhibitors was 0.00144 ± 0.00012 μm 2 / s, compared with the diffusion coefficient of CVS-11 in untreated nerve cells, its movement rate is about half. The experimental results directly visualize the inhibitory effect of the inhibitor, proving that RABV labeled with AIE materials can be used to screen antiviral drugs and visualize their effects.

[0124] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing an AIE material for labeling rabies virus, characterized in that: The steps include: 1mmol TPE-BP-cho was dissolved in 20mL tetrahydrofuran, and a 0.5M ethylmagnesium bromide solution containing 1.2mmol THF was added under N2 atmosphere, and stirred at room temperature overnight; saturated ammonium chloride solution was used for quenching, the crude product was extracted with dichloromethane, and then dried with MgSO4, and the organic layer was concentrated and purified with silica; a mixed solution of n-hexane and dichloromethane with a volume ratio of 2:1 was used for gel chromatography to obtain a solid yellow powder; 119.2mg of the solid yellow powder and 10mmol MnO2 were added to 20mL dichloromethane, stirred at room temperature for 24h under N2 atmosphere, and n-hexane / DCM silica gel chromatography was used to obtain the compound TPE-BP-pyo, which is the AIE material.

2. The AIE material prepared by the preparation method according to claim 1.

3. A method for labeling rabies virus using the AIE material according to claim 2, characterized in that: The steps include: S1. Mix TPE-BP-pyo and dimethyl sulfoxide in a ratio of 1 g: 100 mL to obtain a solution containing AIE material; S2. Mix the purified rabies virus with the solution containing the AIE material obtained in S1 at a volume ratio of 10:1 and combine at room temperature for 1-2 hours.

4. Rabies virus labeled with AIE material obtained by the method of claim 3.

5. Use of the AIE material of claim 2, the method of claim 3, or the AIE material of claim 4 to label rabies virus in real-time tracking of rabies virus in living cells.

6. Use of the AIE material of claim 2, the method of claim 3, or the AIE material of claim 4 labeled with rabies virus in studying the infection mechanism of rabies virus.

7. Use of the AIE material according to claim 2, the method according to claim 3, or the AIE material according to claim 4 labeled with rabies virus in screening anti-rabies virus drugs.

8. The use according to claim 6 or 7, characterized in that: This includes using rabies virus-inoculated cells labeled with AIE materials to perform statistical analysis on the motion trajectories, time-averaged mean square displacement, ensemble-averaged mean square displacement, and displacement probability density distribution of all virus particles in a single cell.

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