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Luciferase labeling system of ectromelia virus and application of luciferase labeling system

A mousepox virus and sequence technology, applied in the field of luciferase labeling system, to achieve the effect of improving the sensitivity of virus monitoring and stabilizing the quality of fluorescence

Active Publication Date: 2014-12-03
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There is no report on Gaussia luciferase tracing of mousepox virus

Method used

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  • Luciferase labeling system of ectromelia virus and application of luciferase labeling system
  • Luciferase labeling system of ectromelia virus and application of luciferase labeling system
  • Luciferase labeling system of ectromelia virus and application of luciferase labeling system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1, construction and identification of LUC-labeled recombinant mousepox virus

[0058] 1. Construction and identification of homologous recombination plasmids

[0059] 1. Construction and identification of recombinant plasmid pGPT-IN

[0060] Positions 89041-89518 (corresponding to positions 1-478 of sequence 1) of the genome DNA sequence of the wild-type mousepox virus Moscow strain (GenBank No. NC_004105.1, Up date: 2012-11-22) were named as upstream same source arm) and positions 89519-90053 (corresponding to positions 479-1013 of sequence 1, named downstream homology arms) were cloned into the upstream and downstream of the gpt gene of plasmid pSV2-gpt, respectively, to obtain the recombinant plasmid pGPT-IN. The specific operation is as follows:

[0061] (1) Extraction of genomic DNA of wild-type mousepox virus Moscow strain

[0062] The specific operation is as follows:

[0063] The wild-type mousepox virus Moscow strain was appropriately diluted with D...

Embodiment 2

[0163] Example 2, Detection of Biological Activity of Recombinant Murine Pox Virus ECTV-LUC

[0164] 1. Determination of one-step growth curve of recombinant mousepox virus ECTV-LUC

[0165] The ECTV-LUC recombinant virus prepared in Example 1 was used to infect (MOI=1) CV-1 cells, after adsorption at 37°C for 1 hour, the virus liquid was removed, and the cells were washed with DMEM for 3 times to remove residual uninfected virus, and then in Cells were cultured at 37°C with DMEM containing 10% (volume percent) FBS. The virus was collected at different time points (0, 2, 4, 6, 8, 10, 12, 24 hours) within 0-24 hours after infection, and the collected virus was subjected to plaque analysis at 37°C to determine the virus titer (PFU / ml ), and then draw a one-step growth curve. At the same time, the wild-type mousepox virus Moscow strain (ECTV-Moscow) was used as a control to detect the difference between the recombinant ECTV-LUC virus and its one-step growth curve. Experiments we...

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Abstract

The invention discloses a luciferase labeling system of an ectromelia virus and an application of the luciferase labeling system. The luciferase labeling system provided by the invention is a recombinant ectromelia virus which is obtained through replacement or insertion of wild ectromelia virus genome DNA. In term of replacement, any one of segments a in the wild ectromelia virus genome DNA is replaced with a segment b; and in term of insertion, the segment b is inserted to any one locus of the segments a in the wild ectromelia virus genome DNA; the segments a are DNA segments shown in a sequence 1 in a sequence list; and the segment b is a DNA segment of a coding gene containing Gaussia luciferase (LUC). The ECTV-LUC can be used for research in parallel with a wild virus. In addition, through the signal amplifying effect of LUC, the ECTV-LUC can greatly enhance the monitoring sensitivity of virus, thus providing a new idea for establishing a novel low-dosage virus subclinical infected animal model and also providing a new technical platform for drug screening of ectromelia virus and other applied researches.

Description

technical field [0001] The invention relates to a mousepox virus luciferase labeling system and its application, in particular to a recombinant mousepox virus obtained by inserting a Gaussia luciferase coding gene into the wild-type mousepox virus Moscow strain genome. Background technique [0002] Mousepox virus (poxvirus of mice) belongs to Poxviridae Chordatepoxvirinae Orthopoxvirus genus, the nucleic acid is double-stranded DNA, has a capsule, and the natural host is mice. If it is higher, it often causes the whole group of mice to be eliminated, which is extremely harmful. The clinical manifestations are characterized by swelling, ulceration, necrosis and even loss of toes in limbs, tail and head, so mousepox virus is also called Ectromelia virus (ECTV). It is closely related to smallpox virus and vaccinia virus in serology and immunology, and is widely used in the world to construct small animal models for screening anti-variola virus drugs. [0003] Bioluminescent t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/85C12Q1/70C12N5/10C12R1/93
Inventor 常国辉刘京梅孙走南杨益苏文莉唐玥何湘
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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