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Method of utilizing rare earth up-conversion fluorescent particles to mark sendai virus envelope

A technology of rare earth up-conversion and fluorescent particles, which is applied in the field of biotechnology and materials, can solve the problems of weak fluorescence of fluorescent proteins, long fusion expression cycle, and great influence on virus particle activity, and achieve high-efficiency expression

Active Publication Date: 2020-02-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method still has certain limitations: the fusion of fluorescent protein and virus particles has a long expression cycle, cumbersome operation steps, and has a great impact on the activity of virus particles, and the fluorescent protein’s autofluorescence is weak and easy to photobleach, which is not conducive to long-term development. Virus tracing; compared with fluorescent proteins, the labeling process of organic fluorescent dyes as virus markers is relatively simple and has little impact on virus activity, but their emission spectrum is wide and their photostability is poor, and long-term real-time fluorescence monitoring is relatively difficult. To a certain extent, its application in the field of single-particle tracing is limited; although quantum dots solve the problem of partial fluorescence quenching, if they are applied to living virus tracing, there are still limitations such as poor penetration of visible light, short fluorescence lifetime, and high background noise. Limitations for studying live infection with viruses

Method used

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  • Method of utilizing rare earth up-conversion fluorescent particles to mark sendai virus envelope
  • Method of utilizing rare earth up-conversion fluorescent particles to mark sendai virus envelope
  • Method of utilizing rare earth up-conversion fluorescent particles to mark sendai virus envelope

Examples

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Effect test

Embodiment 1

[0049] Synthesis of Rare Earth Upconversion Fluorescent Particles by High Temperature Oil Phase Method

[0050] According to the mass fraction ratio, it is yttrium chloride: ytterbium chloride: erbium chloride==78:20:5 raw materials are added in the round-bottomed flask, solvent water is added to make the above-mentioned substances dissolve completely; under magnetic stirring conditions, it is heated to boiling, Until the rare earth salt solution turns into a white solid; after the water is evaporated to dryness, cool to 60°C, add oleic acid:octadecene=2:1 into a round-bottomed flask according to the volume ratio to completely dissolve the white solid; Sodium hydroxide: ammonium fluoride = 1:3, add the two, and add methanol to dissolve completely, the mass ratio of ammonium fluoride to the above-mentioned thulium chloride = 3:1; adjust the temperature to 120°C, and vacuum for 30min , through argon gas; quickly heat up to 300 ° C, and maintain the reaction for 1 h; after the en...

Embodiment 2

[0052] Synthesis of Rare Earth Upconversion Fluorescent Particles by High Temperature Oil Phase Method

[0053] According to the mass fraction ratio, it is yttrium chloride: ytterbium chloride: erbium chloride==78:20:4 raw materials are added in the round bottom flask, solvent water is added to make the above-mentioned substances dissolve completely; under magnetic stirring conditions, it is heated to boiling, Until the rare earth salt solution turns into a white solid; after the water is evaporated to dryness, cool to 60°C, add oleic acid:octadecene=2:1 into a round-bottomed flask according to the volume ratio to completely dissolve the white solid; Sodium hydroxide: ammonium fluoride = 1:3, add the two, and add methanol to dissolve completely, the mass ratio of ammonium fluoride to the above-mentioned thulium chloride = 3:1; adjust the temperature to 120°C, and vacuum for 30min , blowing argon; rapidly raising the temperature to 300° C., and maintaining the reaction for 1 h;...

Embodiment 3

[0055] Synthesis of Rare Earth Upconversion Fluorescent Particles by High Temperature Oil Phase Method

[0056] According to the mass fraction ratio, it is yttrium chloride: ytterbium chloride: erbium chloride==78:20:2 raw materials are added in the round bottom flask, solvent water is added to make the above-mentioned substances dissolve completely; under magnetic stirring conditions, be heated to boiling, Until the rare earth salt solution turns into a white solid; after the water is evaporated to dryness, cool to 60°C, add oleic acid:octadecene=2:1 into a round-bottomed flask according to the volume ratio to completely dissolve the white solid; Sodium hydroxide: ammonium fluoride = 1:3, add the two, and add methanol to dissolve completely, the mass ratio of ammonium fluoride to the above-mentioned thulium chloride = 3:1; adjust the temperature to 120°C, and vacuum for 30min , blowing argon; rapidly raising the temperature to 300° C., and maintaining the reaction for 1 h; af...

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Abstract

The invention discloses a method of utilizing rare earth up-conversion fluorescent particles to mark sendai virus envelope. The method includes following steps: building efficient expression plasmid pCDH-BirA-AP-GPI containing biotin ligase (BirA), biotin binding site (APtag) and glycosylphatidylinositol cytomembrane anchoring sequence (GPI); synthesizing streptavidinized rare earth up-conversionfluorescent particles; preparing enveloped biotinylated sendai virus; utilizing the streptavidinized rare earth up-conversion fluorescent particles to mark the enveloped biotinylated sendai virus. Byadopting virus envelope marking technology, virus marking efficiency of 40-60% can be realized; by excited by near infrared light of 980nm, the rare earth up-conversion fluorescent particles can emitgreen fluorescence of 540nm, and observation can be realized 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 marking the envelope of Sendai virus with rare earth up-conversion fluorescent particles. Background technique [0002] The current virus single particle labeling method mainly includes the application of fluorescein, fluorescent protein and quantum dot nanoparticles to label the envelope, capsid and nucleic acid structure of the virus respectively or simultaneously. However, this method still has certain limitations: the fusion of fluorescent protein and virus particles has a long expression cycle, cumbersome operation steps, and has a great impact on the activity of virus particles, and the fluorescent protein’s autofluorescence is weak and easy to photobleach, which is not conducive to long-term development. Virus tracing; compared with fluorescent proteins, the labeling process of organic fluorescent dyes as virus markers is relati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/66
CPCC12N7/00C12N15/85C12N15/66C12N2760/18851
Inventor 王汉杰潘惠卓常津
Owner TIANJIN UNIV
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