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Ectromelia virus GFP (green fluorescent protein) tracer system and application thereof

A mousepox virus and sequence technology, applied in virus/bacteriophage, microbe-based methods, using vectors to introduce foreign genetic material, etc., to achieve the effect of improving the sensitivity of virus monitoring and stabilizing the quality of fluorescence

Active Publication Date: 2015-07-08
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report on the GFP tracer of mousepox virus

Method used

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  • Ectromelia virus GFP (green fluorescent protein) tracer system and application thereof
  • Ectromelia virus GFP (green fluorescent protein) tracer system and application thereof
  • Ectromelia virus GFP (green fluorescent protein) tracer system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, construction and identification of GFP-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 wit...

Embodiment 2

[0164] Example 2, Detection of Biological Activity of Recombinant Murine Pox Virus ECTV-GFP

[0165] 1. Determination of one-step growth curve of recombinant mousepox virus ECTV-GFP

[0166] The ECTV-GFP recombinant virus prepared in Example 1 was used to infect (MOI=1) CV-1 cells, and 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 ECTV-GFP recombinant virus and its one-step growth curve. Experime...

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Abstract

The invention discloses an ectromelia virus GFP (green fluorescent protein) tracer system and an application thereof. The system provided by the invention is a recombinant ectromelia virus which is the recombinant virus obtained by substitution or insertion of wild type ectromelia virus genomic DNA (deoxyribonucleic acid); the substitution is as follows: any fragment in a fragment a in the wild type ectromelia virus genomic DNA is substituted by a fragment b; the insertion is as follows: the fragment b is inserted at any site in the fragment a in the wild type ectromelia virus genomic DNA; the fragment a is a DNA fragment as shown in sequence 1 in a sequence table; the fragment b is the DNA fragment containing encoding genes of a green fluorescent protein. The ECTV (ectromelia virus)-GFP disclosed by the invention and a wild type virus can be used for research in parallel. In addition, due to the signal amplification effect of the GFP, the ECTV-GFP can greatly improve the virus monitoring sensitivity, provide a new idea for establishing a novel low-dose virus subclinical infection animal model, and provide a new technical platform for screening anti-ectromelia virus medicaments and other application research.

Description

technical field [0001] The invention relates to a GFP tracer system of mousepox virus and its application, in particular to a recombinant mousepox virus obtained by inserting a GFP coding gene into the genome of wild-type mousepox virus Moscow strain. 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] Green fluorescent protein (GFP) is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N5/10C12N1/15C12N1/19C12N1/21C12N15/63A01K67/027C12R1/93
Inventor 刘京梅常国辉孙走南杨益苏文莉唐玥何湘
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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