CN105717084A - Non-enveloped virus quantum dot marking method and application - Google Patents
Non-enveloped virus quantum dot marking method and application Download PDFInfo
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Abstract
The invention discloses a non-enveloped virus quantum dot marking method and application.The method comprises the steps that biotinylation virus suspension is centrifuged, and biotinylation non-enveloped viruses of the uniform structure are obtained; the viruses are inoculated into susceptible cells, quantum dots are added for incubation, and non-enveloped viruses marked with the quantum dots can be obtained.By means of the marked non-enveloped viruses, non-enveloped virus outer capsid protein immunofluorescence marking and cell specific component fluorescent protein positioning, dynamic tracking of a non-enveloped virus living body in the invasion process and positioning of interaction components of the non-enveloped viruses and host cells are achieved.The method can also be used for monitoring the host cell invasion path of the viruses in real time, and is particularly applicable to identifying the interaction components of fish non-enveloped viruses and host cells and also applicable to screening, research and development of fish reovirus resistance preparations.
Description
Technical field
The invention belongs to virusology and aquatic animal medical domain, be specifically related to a kind of without togavirus quantum dot marking method, also relate to the purposes of a kind of quantum dot marking method without togavirus.
Background technology
As the representative without togavirus, reovirus is in icosahedron, has double capsid, the genomic pathogenic virus of the double-stranded RNA containing multi-segmental.The host range of reovirus is extremely wide, people, vertebrates (including Fish), invertebrates and plant can be infected, the virosis that host is serious can be caused, as caused the serious hemorrhagic disease of Fish and high mortality, [Zhang Qiya builds virtue in osmanthus, 2014, Chinese science: life sciences, 2014,44:1236 1252].Poisoning intrusion is the vital first step in its replicative cycle.Without togavirus invade the mode of host cell and process with have togavirus and exist significant difference [Groveetal, J.CellBiol, 2011,195 (7): 1071-1082；Whiteetal, CritRevBiochemMolBiol, 2008；43 (3): 189 219].For efficient prevention and control virosis, cause of disease phagocytic process must be carried out monitor in real time.Utilize new material, set up accurate, stable single virion tracer technique, it is how to break through host's barrier, the dynamic process of invasion cell and viral and host interaction to understanding without togavirus, and then understanding is without the mechanism of causing a disease of togavirus, the research and development new antiviral drugs of series and associated biomolecule preparation are significant.
In recent years, by carrying out quantum dot (Quantumdots, DQs) labelling on the cyst membrane of virus, can to there being togavirus to carry out spike [Zhangetal, Biomaterials, 2013,34:7506-7518；Zhangetal, Theranostics, 2014,4 (3): 307-315].But, owing to lacking cyst membrane without togavirus, the method that there is no directly carries out labelling to without togavirus according to existing method.If individually adopting fluorochrome label, then it is subject to that light resistance is more weak, bio-compatible scope has limitation；And through genetic manipulation fluorescent protein labeling, be then likely to make the activity of virus reduce and even lose.Therefore, advantage [Zhaoetal such as can answering novel nano-material quantum dot high resistance photobleaching, long fluorescence lifetime and wide biocompatibility need to be set up, Chem.Commun.2008,41:5116 5118], it is suitable for again carrying out quantum dot-labeled new technique to without togavirus simultaneously.
Summary of the invention
It is an object of the invention to there are provided one without togavirus quantum dot marking method, this technical mark virion is reliable and stable, and homogeneity is good, it is adaptable to carry out labelling to without togavirus.
Another object of the present invention is to there are provided the application of a kind of quantum dot marking method without togavirus.Quantum dot-labeled is inoculated in the sensitive cells growing up to monolayer on glass bottom capsule without togavirus, then with Green fluorescent dye indicator cells specific components (as with cytoplasma membrane dyestuff CellMaskTMDye cell membrane), put the quantum dot-labeled relative position without togavirus (redness) Yu cell specific components of continuous observation under fluorescence microscope, can the viral movement locus of real-time tracing.
This is a kind of for the high efficiency method without togavirus spike in real time for a long time.Through with outer capsid proteins immunofluorescence label without togavirus test and ImageProPlus computed in software, the method labelling without the efficiency of togavirus more than 75%.
To achieve the above object, the present invention takes techniques below measure:
A kind of without togavirus quantum dot marking method, comprise the steps:
1) without, in togavirus suspension, adding biotin and hatch, carry out the biotinylation without togavirus.
2) ultracentrifugation removes incomplete granule without togavirus and other impurity；First biotinylated viral suspension is centrifuged, collect precipitation, add PBS to suspend, it is centrifuged through discontinuous sucrose density gradient (20%, 30%, 40%, 50% and 60%), collects each centrifugal band respectively, centrifugal after adding PBS washing, collect precipitation, it is resuspended in TE buffer, negative staining electron microscopic observation, it is thus achieved that structure is homogeneous, biotinylated without togavirus.
3) by step 2) biotinylation that obtains is inoculated in permissive cell without togavirus, and add quantum dot and hatch, carry out quantum dot-labeled without togavirus.
In the process described above, it is preferred that described is turbot reovirus (SMReV) without togavirus.
In the process described above, it is preferred that centrifugal speed is 110,000g, centrifuging temperature is 4 DEG C.
A kind of application of quantum dot marking method without togavirus, including the method in monitoring without the application in togavirus infected cell process, or the application that the method is in research is made mutually without togavirus and host cell；Or the application in screening anti-preparation without togavirus.
Concrete, described method is utilized to include without the process of the application in togavirus infected cell process in monitoring: the quantum dot-labeled cell without togavirus inoculation, use cytoplasma membrane dyeing, under fluorescence microscope, the quantum dot-labeled movement locus without togavirus is carried out spike；
The application in research is made from host cell mutually without togavirus of the described method is utilized to include: different cellular components are carried out fluorescence localization first with merging fluorescin or fluorescent probe etc. by cell monolayer, the biotinylation inoculated, without togavirus, carries out quantum dot-labeled.Fix after cell is cultivated different time, carry out fluorescence microscopy observation to quantum dot-labeled without togavirus positioning cells component together, to find out without the togavirus direction of motion, track and the component with cell interaction.
Utilize the application in screening anti-preparation without togavirus of the described method to include: by biotinylation without togavirus or quantum dot-labeled without togavirus and other reagent effects after, whether can be combined with specific cells component, screen the anti-preparation without togavirus.
Based on said method, the present invention is to without togavirus, as the process of fish cell invaded by reovirus, monitoring in real time.By building fusion genes plasmid or fluorescent probe, also can carry out labelling and qualification to the cellular component (receptor) without togavirus effect, especially can recognize that Fish reovirus makes component mutually in host cell.It addition, can pass through at biotinylation without togavirus or quantum dot-labeled without after togavirus and other reagent effects, if can be combined with specific cells component, screen and develop anti-Fish without togavirus preparation.
Compared with prior art, the invention have the advantages that
1. the present invention realizes carrying out quantum dot-labeled without togavirus individual particle, by quantum dot-labeled, can carry out long-time spike in real time to without togavirus granule.
2. the present invention is based on strategy quantum dot-labeled, that combine without togavirus outer capsid proteins immunofluorescence label and cell specific components fluorescin location, adopt ultracentrifugation to separate and substitute desalting column removing unreacting reagent, significantly improve the quality without togavirus to be marked and quantity, with reference to outer capsid proteins labelling, demonstrate quantum dot-labeled has the feature that performance is excellent, efficiency is high without togavirus.
3. utilize the tracing method of the present invention, finely accurate spike can not only invade the track of cell membrane without togavirus；And obtain without togavirus in inoculation 90min, successively with host cell early endosome, late endosomal and lysosome network for location picture altogether, directly perceived displaying without togavirus SMReV is to carry out transporting in kytoplasm through interior body lysosome system.
4. the invention provides a kind of screening the anti-Fish of development without the application in togavirus preparation, be recognize the useful tool researched and developed without togavirus infection mechanism and anti-virus formulation.The present invention also can be widely used in the fields such as aquatic animal medical science.
5. utilizing quantum dot marking method without togavirus provided by the invention, labeling effciency may be up to more than 75%.
Accompanying drawing explanation
Fig. 1 is the SMReV of a kind of quantum dot-labeled and outer capsid proteins immunofluorescence label and in pericellular distribution.
A. the general smooth microgram of individual cells.B.C and D is fluorescence microscopy figure, wherein the quantum dot-labeled SMReV without togavirus of B. (redness)；C. outer capsid proteins immunofluorescence label is without togavirus SMReV (green)；D. being the stacking chart of B and C, overlap (in yellow) signal red, green is defined as quantum dot-labeled without togavirus.Scale: 5 μm.
Fig. 2 is the movement locus of a kind of SMReV without togavirus.
Quantum dot-labeled SMReV without togavirus adsorbs (a shows intact cell) and enters the timing variations (0s, 7s, 11s and 13s, cell membrane partial enlargement) of kytoplasm through cell membrane.Just start quantum dot-labeled SMReV without togavirus and position (a and 0s altogether with cyst membrane, in yellow), extend in time, quantum dot-labeled progressively disengage cell membrane (green) without togavirus (redness), enter in kytoplasm (13s).
Fig. 3 is a kind of quantum dot-labeled SMReV without togavirus (redness) and intracellular lysosome system (green) network for location picture altogether.
Rab5: when 30min, SMReV mainly position altogether with early endosome.Rab7: when 60min, SMReV mainly position altogether with late endosomal；Lysosome: when 90min, SMReV mainly position altogether with lysosome.Scale: 5 μm.
Detailed description of the invention
Technical scheme of the present invention, if not otherwise specified, is the ordinary skill in the art.Described reagent or material, if not otherwise specified, derive from commercial channel.The embodiment of the present invention is with to the labeling method of SMReV with should be used for being expanded on further, but described methods and applications are not limited to SMReV.
Embodiment 1:
The labeling method of quantum dot without togavirus and labeling effciency
1) SMReV amplification cultivation:
According to a conventional method, Ctenopharyngodon idellus fin ray cell line (GCF) [Keetal.BMCGenomics2011,12:323] being carried out Secondary Culture, after 16h, cell grows up to monolayer, every 25cm2Culture bottle cell inoculates 100 μ l turbot reovirus (SMReV) stock solutions, is placed in 20 DEG C of cultivations, collects sick cell, multigelation 3 times after 7 days.4 DEG C of difference (4,000g and 12,000g) centrifugal 20min, collects supernatant, it is thus achieved that turbot reovirus SMReV suspension.
2) containing 107In the SMReV suspension of the 300mL of individual RNA copy, add biotin (sulfo-NHS-LC-biotin, Thermo) the solution 300 μ L that concentration is 0.01mg/ μ l, be placed in 25 DEG C of shaking tables, react 2h.With 110,000g (BeckmanSW41 rotary heads), 4 DEG C, centrifugal 1.5h, collection precipitation, addition PBS (137mMNaCl, 2.7mMKCl, 8.1mMNa2HPO4,1.5mMKH2PO4, pH7.5) buffer suspends；Through discontinuous sucrose density gradient (20%, 30%, 40%, 50% and 60%), with 110,000g, 4 DEG C, centrifugal 1.5h；Collect the virus of each layer respectively, after adding PBS washing, 110,000g centrifugal 1.5h, collect precipitation；It is suspended from TE (10mMTris-Cl, 1mMEDTA, the pH7.4) buffer of 10mL, take viral suspension negative staining sample preparation, Electronic Speculum (JEM1230) is observed, choose that particle shape is homogeneous, structural integrity and a fairly large number of viral suspension standby, it is thus achieved that biotinylation is without togavirus.
3) by GCF passage in 35mm glass bottom capsule, after cultivating 16h, cell grows up to monolayer, remove supernatant, inoculate that 100 μ l are biotinylated to be placed in 4 DEG C without togavirus and hatch the 30min PBS containing 0.1% Ox blood serum 3 times, add the 5nM of the 100 μ L quantum dot (605nmDQs, Invitrogen) with Avidin；Hatch 15min for 4 DEG C, wash 3 times with the PBS containing 0.1% Ox blood serum.Select 561nm laser beam to excite DQs605 (taking on a red color), use 617/73 optical filter, observe under high-rate laser Laser Scanning Confocal Microscope, obtain quantum dot-labeled without togavirus.
4) fix through 4% paraformaldehyde, the mouse resisting anteserum (anti-VP7) prepared by outer capsid proteins gene expression product is encoded as primary antibodie with SMReV, immunofluorescence dyeing (in green) is carried out again to without togavirus, fluorescence microscopy Microscopic observation (see Fig. 1), overlap (in yellow) signal red, green is defined as quantum dot-labeled without togavirus.Through ImageProPlus computed in software, the percent of yellow fluorescence signal and the ratio of red fluorescent is labeling effciency, quantum dot institute labelling without the efficiency of togavirus more than 75%.
Embodiment 2:
A kind of quantum dot marking method without togavirus is without the application in the invasion spike of togavirus
1) by embodiment 1 step 3) in be vaccinated with the quantum dot-labeled cell monolayer without togavirus, with cytoplasma membrane dyestuff (CellMaskTM, Invitrogen) dye；
2) with being placed in the culture systems that high-rate laser Laser Scanning Confocal Microscope is subsidiary by capsule, carry out cultivating online and Fluirescence observation；
3) select 561nm laser beam to excite DQs605, observe with 617/73 optical filter quantum dot-labeled without togavirus (redness)；640nm laser beam is selected to excite CellMaskTM, with 685/40nm optical filter observation of cell film (green)；
4) continuous or separated in time is taken pictures, utilize image software obtain single particle motion trajectory without togavirus and position the image series (Fig. 2) of (yellow) or relative position in the corresponding time with cell membrane altogether, SMReV can be obtained in real time and invade the movement locus of GCF cell.
Result is as shown in Figure 2: quantum dot-labeled SMReV without togavirus adsorbs (a shows intact cell) and enters the timing variations (0s, 7s, 11s and 13s, cell membrane partial enlargement) of kytoplasm through cell membrane.Just start quantum dot-labeled SMReV without togavirus and position (a and 0s altogether with cyst membrane, in yellow), extend in time, quantum dot-labeled progressively disengage cell membrane (green) without togavirus (redness), enter in kytoplasm (13s).
Embodiment 3:
Without togavirus space-time spike in intracellular lysosome system
1) in the capsule of multiple glass bottoms, Secondary Culture GCF cell, to growing up to monolayer；
2) transfect pRFP-Rab5 plasmid markers early endosome respectively, transfect pRFP-Rab7 plasmid markers late endosomal, with green fluorescence probe dye (LysoTrackerGreen, Invitrogen) labelling lysosome；
3) step 2 in embodiment 1 is inoculated) the middle biotinylated SMReV without togavirus obtained, carry out labelling with quantum dot DQs705 (Ka source, Wuhan) to without togavirus SMReV；
4) after each capsule being cultivated different time (30min, 60min and 90min), respectively with the fixing cell of the paraformaldehyde of 4%；
5) select 488nm laser beam to excite DQs705, observe with 685/40nm optical filter quantum dot-labeled without togavirus (red pseudo-color)；561nm laser beam is selected to excite pRFP-Rab5 and pRFP-Rab7 fluorescence, with body in 617/73 optical filter observation of cell and lysosome (green pseudo-color)；
6) obtain series different time and position (yellow) image altogether without togavirus granule and cellular component.
7) SMReV main and early endosome (Rab5) location altogether when 30min, main and late endosomal (Rab7) location altogether when 60min, main and lysosome (Lysosome) location (see Fig. 3) altogether during 90min.Show and carry out transporting in kytoplasm through interior body lysosome system without togavirus SMReV.Complete without the space-time spike in host cell of the togavirus course of infection.
Result is as shown in Figure 3: Rab5: when 30min, SMReV are main to be positioned altogether with early endosome.Rab7: when 60min, SMReV mainly position altogether with late endosomal；Lysosome: when 90min, SMReV mainly position altogether with lysosome.Scale: 5 μm.
Embodiment 4:
The application in Fish reovirus specific antibody screens of a kind of quantum dot marking method without togavirus:
1) with Fish reovirus SMReV immune animal, SMReV antiserum is prepared, and standby with PBS serial dilution (1:4,1:16,1:64,1:128,1:256) SMReV antiserum；
2) by step 1 in embodiment 3) prepare multiple length and have the capsule of monolayer GCF cell；
3) by step 2 in embodiment 3) described in step pRFP-Rab5 labeled cell early endosome；
4) by step 2 in embodiment 1) obtain biotinylated SMReV, and respectively with the SMReV antiserum of PBS serial dilution or after hatching without sero-fast PBS (comparison), be respectively inoculated in the long capsule having cell monolayer；
5) again by step 3 in embodiment 3), 4) and 5), SMReV is carried out labelling and cultivate 30min, cell is fixed and Fluirescence observation.
In nonreactive serum control capsule, quantum dot-labeled SMReV can position altogether with cell early endosome, but in the antiserum capsule of high concentration (if dilution factor is 1:4 and 1:16), quantum dot-labeled SMReV but can not position with cell early endosome altogether, it was shown that there is the potent antibodies of cell early endosome in antiserum.Accordingly, the specific antibody made mutually for Fish reovirus and cell early endosome can be screened.
Claims (10)
1., without a togavirus quantum dot marking method, comprise the steps:
1) without, in togavirus suspension, adding biotin and hatch, carry out the biotinylation without togavirus；
2) ultracentrifugation removes incomplete granule without togavirus and other impurity；First biotinylated viral suspension is centrifuged, collect precipitation, add PBS to suspend, through discontinuous sucrose density gradient centrifugation, collect each centrifugal band respectively, centrifugal after adding PBS washing, collect precipitation, it is resuspended in TE buffer, negative staining electron microscopic observation, it is thus achieved that structure is homogeneous, biotinylated without togavirus；
3) by step 2) biotinylation that obtains is inoculated in permissive cell without togavirus, and add quantum dot and hatch, carry out quantum dot-labeled without togavirus.
2. method according to claim 1, it is characterised in that: described discontinuous sucrose density is 20%, 30%, 40%, 50% and 60%.
3. method according to claim 1, it is characterised in that: described is turbot reovirus without togavirus.
4. method according to claim 1, it is characterised in that: described centrifugal speed is 110,000g, and centrifuging temperature is 4 DEG C.
5. the method described in claim 1 is being monitored without the application in togavirus infected cell process.
6. the application in research is made mutually without togavirus and host cell of the method described in claim 1.
7. the application in screening anti-preparation without togavirus of the method described in claim 1.
8. application according to claim 5, its application process includes: quantum dot-labeled inoculating cell without togavirus, uses cytoplasma membrane dyeing, under fluorescence microscope, the quantum dot-labeled movement locus without togavirus is carried out spike；
Application according to claim 6, its application process includes: different cellular components are carried out fluorescence localization first with merging fluorescin or fluorescent probe by cell monolayer, and the biotinylation inoculated is without togavirus, then carries out quantum dot-labeled.
9. fix after cell is cultivated different time, carry out fluorescence microscopy observation to quantum dot-labeled without togavirus positioning cells component together, to find out without the togavirus direction of motion, track and the component with cell interaction.
10. application according to claim 7, its application process includes: by quantum dot-labeled without after togavirus and other reagent effects, if can be combined with specific cells component, screen the anti-preparation without togavirus.
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Cited By (2)
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| CN110174387A (en) * | 2019-06-14 | 2019-08-27 | 郑州大学 | A kind of application of fluorescent carbon point in naturally targeting lysosome |
| CN112611742A (en) * | 2021-01-11 | 2021-04-06 | 佛山市第一人民医院（中山大学附属佛山医院） | Zika virus visual marking strategy utilizing photo-click bioorthogonal reaction |
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| CN112611742A (en) * | 2021-01-11 | 2021-04-06 | 佛山市第一人民医院（中山大学附属佛山医院） | Zika virus visual marking strategy utilizing photo-click bioorthogonal reaction |
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