In-vivo imaging tracing system of vaccinia virus and application of system
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A vaccinia virus vector, vaccinia virus technology, applied in the direction of virus/phage, microorganism-based method, using vector to introduce foreign genetic material, etc., to achieve the effect of stable fluorescence quality
Active Publication Date: 2013-09-18
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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[0005] There is no report on Gaussia luciferase tracing of vaccinia virus
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Embodiment 1
[0056] Example 1, construction and identification of LUC-labeled recombinant vaccinia virus
[0057] 1. Construction and identification of homologous recombination plasmids
[0058] 1. Construction and identification of recombinant plasmid pGPT-IN
[0059] Positions 80267-80725 (corresponding to positions 1-459 of sequence 1) of the genomic DNA sequence of the wild-type vaccinia virus WR strain (GenBank number: NC_006998.1, Update: 2012-11-22) were named as the upstream homology arm ) and positions 80726-81194 (corresponding to positions 460-928 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:
[0060] (1) Extraction of genomic DNA of wild-type vaccinia virus WR strain
[0061] The specific operation is as follows:
[0062] The wild-type vaccinia virus WR strain was appropriately diluted with DMEM com...
Embodiment 2
[0163] Example 2, Detection of Biological Activity of Recombinant Vaccinia Virus V.V.-LUC
[0164] 1. Determination of one-step growth curve of recombinant vaccinia virus V.V.-LUC
[0165]Infect (MOI=1) CV-1 cells with the V.V.-LUC recombinant virus prepared in Example 1. After adsorption at 37°C for 1 hour, remove the virus liquid and wash the cells with DMEM for 3 times to remove residual uninfected virus. 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 V.V.-WR was used as a control to detect the difference between the V.V.-LUC recombinant virus and its one-step growth curve. Experiments were repeated three times.
[0166] The results of the determination ...
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Abstract
The invention discloses an in-vivo imaging tracing system of a vaccinia virus and an application of the system. The system disclosed by the invention is a recombinant vaccinia virus which is a recombinant virus obtained by subjecting wild-type vaccinia virus genome DNA (Deoxyribonucleic Acid) to replacement or insertion, wherein the replacement is to replace any segment of a segment a in the wild-type vaccinia virus genome DNA by a segment b; the insertion is to insert the segment b at any locus of the segment a in the wild-type vaccinia virus genome DNA; the segment a is the DNA segment shown in the sequence 1 in a sequence table; and the segment b is the DNA segment containing Gaussia luciferase encoding gene. The V.V. (Vaccinia Virus)-LUC (Luciferase) disclosed by the invention can be applied to a research in parallel with a wild-type virus; and in addition, by virtue of the signal amplification effect of the Gaussia luciferase, the V.V.-LUC can greatly improve the virus monitoring sensitivity, provide a new concept for establishing a novel low-dosage virus subclinical infection animal model, and provide a new technical platform for the application researches on screening of anti-V.V medicines and the like.
Description
technical field [0001] The invention relates to a vaccinia virus live imaging tracing system and application thereof, in particular to a recombinant vaccinia virus obtained by inserting a Gaussia luciferase gene into the wild-type vaccinia virus WR strain genome. Background technique [0002] Vaccinia virus (vaccinia virus) belongs to poxvirus (Poxvirus). Serologically and immunologically, it is closely related to smallpox virus and vaccinia virus, and is used as an antigen for smallpox vaccines. Virus particles have a volume of about 300×230×100 nanometers, an oval brick shape, and a molecular weight of 160-170×10 6 DNA. When people are vaccinated, local lesions usually appear on the skin, but when the immune system is defective, they invade the whole body (generalized acne, vaccina generalisata, also known as progressive post-vaccination eczema), often fatal, and rarely appear in the brain after vaccination inflammation. [0003] Bioluminescent tracer technology is a n...
Claims
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