Antiviral actions, implementation method and use of novel antiviral molecule DRP
A kind of use, virus technology, applied in the fields of biotechnology and medicine, can solve the problems that the research of immune function has not yet waited for
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Embodiment 1
[0118] Example 1: Stimulation of DRP on the production of IFN-β and interferon-inducible genes in the macrophage cell line RAW264.7 into effect
[0119] First, pcDNA3.1 (purchased from Invitrogen) was used as a eukaryotic expression vector, and the cDNA product of DRP with BamHI / KpnI restriction sites was inserted into the corresponding position of the vector, and amplified in E. Coli strain DH-5α The vector is purified to obtain the eukaryotic expression vector of DRP after sequencing identification. Stable transfection (transfection reagent JetPei was purchased from Polyplus company) mouse RAW264.7 cells (purchased from ATCC), and then the resulting stable transfected cell line (1 × 10 5 cells / ml RPMI 1640 medium), treated with 100ng / ml LPS (also known as lipopolysaccharide, which is the ligand of TLR4, purchased from Sigma Company) for different time, collected cells and culture supernatant, prepared cDNA for quantitative RT -PCR (94°C, 30Sec; 58°C, 30Sec; 72°C, 30Sec;...
Embodiment 2
[0123] Example 2: Overexpression of DRP inhibits the replication and amplification of VSV virus in RAW264.7 cells
[0124] Live VSV (gifted by Professor Shu Hongbing, School of Life Sciences, Wuhan University) and UV-inactivated VSV (VSV-UV) at 0.1 MOI were added to RAW264.7 cells, cultured for 12 hours, and the cells and culture supernatant were collected. Among them, the cells were used to detect the mRNA level of the VSV L gene, and the supernatant was used for the plaque formation experiment (PFU, refer to the literature Xu, L.G. et al. Mol. Cell. 19:727-740 (2005)).
[0125] Test results such as image 3 shown. The results showed that stable overexpression of DRP could significantly inhibit the replication of VSV virus in RAW264.7 cell line and the release of VSV virus into the supernatant.
[0126]The results indicated that overexpression of DRP could inhibit the replication and amplification of VSV virus in macrophage cell lines.
Embodiment 3
[0127] Example 3: Inhibitory effect of DRP adenovirus intravenous injection on the amplification of VSV virus in the liver
[0128] First, pShuttle2 in the Adeno-X virus vector system (purchased from Clontech Company) was used as the cloning vector, and the cDNA of DRP was inserted between the SalI and XhoI restriction sites, and the shuttle plasmid was recombined with the viral DNA in HEK293 cells, and amplified. After the amplification, the adenovirus purification kit (purchased from Clontech Company) was used. 1×10 7 PFU Ad-DRP was injected into the tail vein of mice (6-week-old male SDF-grade C57BL6 mice, purchased from Sipper BK Company), and 48 hours later, VSV virus (5×10 6 PFU / mouse). After 12 or 24 hours, the mouse liver was collected, and the lysate was applied to the plaque formation experiment (PFU, performed with reference to the literature Ciavarra, R.P. et al. Virology. 342: 177-189 (2005)).
[0129] Test results such as Figure 4 shown. The results showe...
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