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Non-enveloped virus quantum dot marking method and application

A labeling method and non-encapsulated technology, which is applied in the fields of virology and aquatic animal medicine, can solve the problems of loss, weak light resistance, and reduced virus activity, and achieve high efficiency, improved quality and quantity, and excellent performance.

Pending Publication Date: 2016-06-29
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of envelopes of non-enveloped viruses, it is not yet possible to directly label non-enveloped viruses according to existing methods
If fluorescent dyes are used alone for labeling, the light resistance is weak and the range of biocompatibility is limited; however, fluorescent protein labeling by genetic manipulation may reduce or even lose the activity of the virus

Method used

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  • Non-enveloped virus quantum dot marking method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Labeling method and labeling efficiency of non-enveloped virus quantum dots

[0034] 1) SMReV amplification culture:

[0035] According to conventional methods, the grass carp fin ray cell line (GCF) [Keetal.BMCGenomics2011,12:323] was subcultured, and the cells grew into a monolayer after 16h, and each 25cm 2 Cells in the culture flask were inoculated with 100 μl of turbot reovirus (SMReV) stock solution, and cultured at 20°C. After 7 days, the diseased cells were collected and frozen and thawed three times. The supernatant was collected by centrifuging at 4° C. with differential (4,000 g and 12,000 g) for 20 min to obtain a suspension of turbot reovirus SMReV.

[0036] 2) in incl. 10 7Add 300 μL of sulfo-NHS-LC-biotin (Thermo) solution at a concentration of 0.01 mg / μl to 300 mL of SMReV suspension containing 1 RNA copy, place on a shaker at 25° C., and react for 2 h. Centrifuge at 110,000g (BeckmanSW41 rotor) at 4°C for 1.5h, collect the precipitate, add PBS (137mM...

Embodiment 2

[0040] Application of a non-enveloped virus quantum dot labeling method in the invasion trace of non-enveloped virus

[0041] 1) The monolayer cells inoculated with the quantum dot-labeled non-enveloped virus in Step 3) of Example 1 were stained with a cytoplasmic membrane dye (CellMask TM , Invitrogen) for staining;

[0042] 2) Then place the small dish in the culture system attached to the high-speed laser confocal microscope for online culture and fluorescence observation;

[0043] 3) Choose 561nm laser beam to excite DQs605, use 617 / 73 filter to observe quantum dot-labeled non-enveloped virus (red); choose 640nm laser beam to excite CellMask TM , use a 685 / 40nm filter to observe the cell membrane (green);

[0044] 4) Take pictures continuously or at regular intervals, and use image software to obtain a series of images of the trajectory of a single non-enveloped virus particle and its colocalization (yellow) or relative position with the cell membrane at the correspondin...

Embodiment 3

[0047] Spatiotemporal Tracing of Non-enveloped Viruses in the Endosome-Lysosome System

[0048] 1) Subculture GCF cells in multiple glass-bottomed dishes until they grow into a monolayer;

[0049] 2) Transfect the pRFP-Rab5 plasmid to mark the early endosome, transfect the pRFP-Rab7 plasmid to mark the late endosome, and use the green fluorescent probe dye (LysoTrackerGreen, Invitrogen) to mark the lysosome;

[0050] 3) re-inoculate the biotinylated non-enveloped virus SMReV obtained in step 2) of Example 1, and use quantum dot DQs705 (Wuhan Jiayuan) to label the non-enveloped virus SMReV;

[0051] 4) After incubating each small dish for different time (30min, 60min and 90min), fix the cells with 4% paraformaldehyde respectively;

[0052] 5) Choose 488nm laser beam to excite DQs705, use 685 / 40nm filter to observe and observe quantum dot-labeled non-enveloped virus (red false color); choose 561nm laser beam to excite pRFP-Rab5 and pRFP-Rab7 fluorescence, use 617 / 73 filter Lig...

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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 [0001] The invention belongs to the fields of virology and aquatic animal medicine, and in particular relates to a non-enveloped virus quantum dot marking method, and also relates to an application of the non-enveloped virus quantum dot marking method. Background technique [0002] As a representative of non-enveloped viruses, reovirus is a pathogenic virus with icosahedral shape, double-layer capsid, and multi-segment double-stranded RNA genome. Reoviruses have a wide range of hosts and can infect humans, vertebrates (including fish), invertebrates and plants, and can cause severe viral diseases in the host, such as severe hemorrhagic disease and high mortality in fish [Zhang Qiya & Gui Jianfang, 2014, Chinese Science: Life Science, 2014, 44:1236–1252]. Virus invasion is the crucial first step in its replication cycle. There are significant differences between non-enveloped viruses and enveloped viruses in the way and process of invading host cells [Grovee...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6439
Inventor 张奇亚刘佳
Owner INST OF AQUATIC LIFE ACAD SINICA
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