GFP (Green Fluorescent Protein) tracing system of mouse hepatitis coronavirus and application of tracing system
A coronavirus and hepatitis technology, applied in the field of GFP tracer system of mouse hepatitis coronavirus, to achieve the effect of stable fluorescence quality
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Embodiment 1
[0066] The construction of the recombinant murine hepatitis coronavirus of embodiment 1, GFP mark
[0067] 1. Construction of Homologous Recombination Plasmids
[0068] 1. Construction and identification of recombinant plasmid pGPT-IN-ORF4
[0069] No. 27500-27966 (corresponding to No. 1-467 of Sequence 1) of the genomic cDNA sequence of wild-type murine hepatitis coronavirus A59 strain (GenBank number: NC_001846.1, Up date: 2012-8-23) was named upstream homology arm) and positions 28265-28700 (corresponding to positions 766-1201 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- ORF4. The specific operation is as follows:
[0070] (1) Extraction of vaccinia virus inf-1 genomic DNA
[0071] Since the vaccinia virus vector vaccinia virus inf-1 (referred to as v.v.-inf-1) contains the full-length cDNA of the genome of the wild-type mouse hepa...
Embodiment 2
[0184] Embodiment 2, the biological activity detection of MHV-GFP recombinant virus
[0185] 1. Determination of one-step growth curve of MHV-GFP recombinant virus
[0186]The MHV-GFP recombinant virus prepared in Example 1 was used to infect (MOI=1) 17Cl-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 33°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 33°C to determine the virus titer (PFU / ml ), and then draw a one-step growth curve. At the same time, the wild-type MHV A59 strain was used as a control to detect the difference between the MHV-GFP recombinant virus and its one-step growth curve. Experiments were repeated three times.
[0187] The re...
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