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Construction method of rare earth up-conversion nano-probe labeled virus

A rare earth up-conversion and nano-probe technology, applied in the field of biotechnology and materials, can solve the problems of unfavorable long-term tracing, poor photostability, high background noise, etc., and achieve the effect of high scientific research application prospects

Pending Publication Date: 2020-07-03
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the methods of labeling viruses mainly use fluorescent proteins, organic dyes and quantum dot nanoparticles, but these labeling methods have certain limitations: for example, the expression cycle of fluorescent proteins is long, the operation steps are cumbersome, and the activity of the labeled virus is greatly affected. Moreover, fluorescent proteins have weak autofluorescence and are prone to photobleaching, which is not conducive to long-term tracing; organic fluorescent dyes have wide emission spectra and poor photostability, making it difficult to monitor real-time fluorescence for a long time; when quantum dots are used for live virus tracing, There are still limitations such as poor penetration of visible light, short fluorescence lifetime and high background noise

Method used

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  • Construction method of rare earth up-conversion nano-probe labeled virus
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  • Construction method of rare earth up-conversion nano-probe labeled virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of envelope biotinylated influenza virus

[0031] MDCK cells were cultured in a cell culture dish with a diameter of 10 cm, DSPE-PEG2000-Biotin reagent (final concentration 1 μg / ml) was added to the medium, and the culture dish was placed in a 37°C carbon dioxide incubator for 18-24h. When the cell density in the culture dish grows to 80-90%, discard the medium, wash the cells twice with serum-free medium to remove residual serum, add 5ml serum-free medium and 10μl influenza virus concentrate, mix well and place Incubate in a carbon dioxide incubator at 37°C for 2 hours, shaking the culture dish every half hour. After incubation, the supernatant was discarded, and the complete medium containing 2% FBS and DSPE-PEG2000-Biotin was added. Place the culture dish in a 37°C carbon dioxide incubator for 48-72h. When 50% of the cells died, the cells and supernatant were collected in a centrifuge tube, and the cells were completely broken up by repeated freezing an...

Embodiment 2

[0033] Preparation of envelope biotinylated influenza virus

[0034] MDCK cells were cultured in a cell culture dish with a diameter of 10 cm, DSPE-PEG2000-Biotin reagent (final concentration 5 μg / ml) was added to the medium, and the culture dish was placed in a 37°C carbon dioxide incubator for 18-24h. When the cell density in the culture dish grows to 80-90%, discard the medium, wash the cells twice with serum-free medium to remove residual serum, add 5ml serum-free medium and 10μl influenza virus concentrate, mix well and place Incubate in a carbon dioxide incubator at 37°C for 2 hours, shaking the culture dish every half hour. After incubation, the supernatant was discarded, and the complete medium containing 2% FBS and DSPE-PEG2000-Biotin was added. Place the culture dish in a 37°C carbon dioxide incubator for 48-72h. When 50% of the cells died, the cells and supernatant were collected in a centrifuge tube, and the cells were completely broken up by repeated freezing an...

Embodiment 3

[0036] Preparation of envelope biotinylated influenza virus

[0037] MDCK cells were cultured in a cell culture dish with a diameter of 10 cm, DSPE-PEG2000-Biotin reagent (final concentration: 50 μg / ml) was added to the culture medium, and the culture dish was placed in a 37°C carbon dioxide incubator for 18-24h. When the cell density in the culture dish grows to 80-90%, discard the medium, wash the cells twice with serum-free medium to remove residual serum, add 5ml serum-free medium and 10μl influenza virus concentrate, mix well and place Incubate in a carbon dioxide incubator at 37°C for 2 hours, shaking the culture dish every half hour. After incubation, the supernatant was discarded, and the complete medium containing 2% FBS and DSPE-PEG2000-Biotin was added. Place the culture dish in a 37°C carbon dioxide incubator for 48-72h. When 50% of the cells died, the cells and supernatant were collected in a centrifuge tube, and the cells were completely broken up by repeated f...

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Abstract

The invention discloses a construction method of a rare earth up-conversion nano-probe labeled virus. The construction method comprises the following steps: 1) preparation of an enveloped biotinylatedinfluenza virus; 2) synthesis of a streptavidin-based rare earth up-conversion nano-probe; and 3) using the streptavidin-based rare earth up-conversion nano-probe to label the enveloped biotinylatedvirus. The prepared rare earth up-conversion nano-probe can be combined with an enveloped biotinylated Sendai virus to realize labeling of the viral envelope. The virus envelope labeling technology ofthe invention can realize the virus labeling efficiency greater than 90%. The rare earth up-conversion nano-probe can emit green fluorescence with a wavelength of 540 nm under the excitation of 980 nm near infrared light, and can be used for observation under a confocal microscope.

Description

technical field [0001] The invention belongs to the field of biotechnology and material technology, and in particular relates to a method for constructing a rare earth upconversion nano-labeled virus. Background technique [0002] At present, the methods of labeling viruses mainly use fluorescent proteins, organic dyes and quantum dot nanoparticles, but these labeling methods have certain limitations: for example, the expression cycle of fluorescent proteins is long, the operation steps are cumbersome, and the activity of the labeled virus is greatly affected. Moreover, fluorescent proteins have weak autofluorescence and are prone to photobleaching, which is not conducive to long-term tracing; organic fluorescent dyes have wide emission spectra and poor photostability, making it difficult to monitor real-time fluorescence for a long time; when quantum dots are used for live virus tracing, However, there are still limitations such as poor penetration of visible light, short f...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02C09K11/02
CPCC12N7/00C09K11/025C12N2760/16151
Inventor 常津庞高举王汉杰张英英潘惠卓
Owner TIANJIN UNIV
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