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GFP (Green Fluorescent Protein) tracing system of vaccinia virus and application of GFP tracing system
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A technology of vaccinia virus and vaccinia virus vector, which is applied in the application, virus/phage, introduction of foreign genetic material using vectors, etc., to achieve the effect of stable fluorescence quality and improving the sensitivity of virus monitoring
Active Publication Date: 2014-12-03
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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[0004] There is no report on GFP tracing of vaccinia virus
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Embodiment 1
[0056] Embodiment 1, construction and identification of GFP-labeled recombinant vaccinia virus
[0057] 1. Construction and identification of homologous recombination plasmids
[0058] 1. Construction and identification of recombinant plasmid pGPT-IN
[0059] No. 80267-80725 (corresponding to No. 1-459 of Sequence 1) of the genomic DNA sequence of wild-type vaccinia virus WR strain (GenBank number: NC_006998.1, Up date: 2012-11-22) was named upstream homolog 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 complete medium, added to...
Embodiment 2
[0166] Example 2, Detection of Biological Activity of Recombinant Vaccinia Virus V.V.-GFP
[0167] 1. Determination of one-step growth curve of recombinant vaccinia virus V.V.-GFP
[0168]Infect (MOI=1) CV-1 cells with the V.V.-GFP recombinant virus prepared in Example 1. 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. 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, wild-type V.V.-WR was used as a control in the experiment to detect the difference between the V.V.-GFP recombinant virus and its one-step growth curve. Experiments were repeated three times.
[0169] The re...
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Abstract
The invention discloses a GFP (Green Fluorescent Protein) 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 into 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 GFP encoding gene. The V.V. (Vaccinia Virus)-GFP 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 GFP, the V.V. -GFP 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 GFP tracer system of vaccinia virus and its application, in particular to a recombinant vaccinia virus obtained by inserting a GFP coding gene into the genome of wild-type vaccinia virus WR strain. 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] Green fluorescent protein (GFP) is a protein with uniqu...
Claims
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