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Method for marking live viral particles in multicolor

A virus particle and multi-color labeling technology, applied in the interdisciplinary field, can solve the problems of loss and reduction of virus infection ability

Active Publication Date: 2013-10-09
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the virus is dissociated and labeled by external force, the infection ability of the virus will be greatly reduced or even completely lost.

Method used

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  • Method for marking live viral particles in multicolor
  • Method for marking live viral particles in multicolor
  • Method for marking live viral particles in multicolor

Examples

Experimental program
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Embodiment 1

[0049] A method for intracellular two-color fluorescent labeling of living virus particles based on virus self-assembly system, metal complexes and genetic engineering technology, the steps are as follows:

[0050] (1) With the help of a virus display system, the green fluorescent protein (EGFP) was fused with the baculovirus envelope protein GP64 to construct a recombinant baculovirus with green fluorescent protein on GP64. According to the AcMNPV genome (GenBank: L22858.1) sequence in GenBank, the primer design software Primerpremier 5 (Premier Company) was used to design EGFP / gp64 gene expression primers ( Image 6 ), EcoR I and HindIII restriction sites were added at the 5' and 3' of gp64, respectively. First, use the purified pEGFP-N1 plasmid (Clonetech) as a template, FEG1 and REG-G as primers for PCR amplification, use the obtained PCR product EGP0 as a template, and use FN2 and REG-G as primers for PCR amplification again , get a fragment containing GP64 signal peptide...

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Abstract

The invention discloses a method for marking live viral particles in multicolor, which comprises the steps of: A, with help of a virus showing system, executing fusion expression of green fluorescent protein and virus envelope protein GP64, and constructing recombinant viruses with the green fluorescent protein on the GP64; B, adding metal complex [Ru(phen)2(dppz)]2+ solution into Grace's culture medium of fetal calf serum to cultivate sf host cells; adding the constructed recombinant viruses Bac-EGFP into the cultivated host cells for virus infection; putting the recombinant viruses and the host cells in a cultivation box for cultivation, and harvesting progeny viruses; and C, collecting, purifying progeny virus particles marked in two colors, treating the obtained progeny viruses through a transmission electron microscope, expressing structures of the progeny virus particles through inductive coupling plasma-mass spectrometry and a laser con-focal scanning microscope; measuring content of the metal complex in each single virus particle; and showing dual fluorescent signals of the single virus particle. The method disclosed by the invention is simple and convenient in operation, and high in marking efficiency, and can obtain marked viruses with excellent fluorescent signals, and satisfy the demands of researches about tracing the live virus particles to infect the host cells in real time in a better way.

Description

technical field [0001] The invention relates to the interdisciplinary fields of biology, chemistry, materials, medicine, virology and other disciplines, and more specifically relates to a method for multicolor labeling living virus particles. Background technique [0002] Real-time tracking of live virus particles infecting host cells is of great significance for in-depth understanding of virus infection mechanisms and early treatment of virus-induced diseases. In previous studies, researchers mainly used two methods to label the virus and use it for tracer research: 1) fusion of a fluorescent protein in the outer protein region of the virus; 2) direct fluorescent molecular probes through chemical reactions Conjugates to viral proteins. These methods can provide a lot of information about the endocytosis of viruses into host cells and the fusion of enveloped viruses with host cell membranes. However, virus infection of host cells is an extremely complex process, the main p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N7/02C12N15/62C12N15/866
Inventor 何治柯王汉中周鹏
Owner WUHAN UNIV