Signal for packaging of influenza virus vectors
A technology of influenza virus and carrier, which is applied in the direction of virus/bacteriophage, introduction of foreign genetic material using carrier, recovery/purification, etc., which can solve the problem of inability to maintain stable virus particles
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0106] Materials and Methods
[0107] virus and cell . Human H3N2 virus isolated from a single patient, localized in embryonated eggs (A / Tottori / AT1 / AM2AL3 / 94; AM1AL3) or madin-darby canine kidney, was obtained from T.ito (Tottori University, Tottori, Japan). Virus stocks were cultured in 10-day-old embryonated eggs (AMZAL3 virus) or MDCK cells in Minimal Essential Medium (MEM) supplemented with 0.3% bovine serum albumin and 0.5 mg trypsin / ml (K4 virus) cultivated. MDCK cells were cultured in MEM supplemented with 5% newborn bovine serum (Sigma, St. Louis, MO).
[0108] Generation of lectin-resistant cell lines. MDCK cells grown to 75% confluence were washed three times with phosphate-buffered saline, and mixed with Korean Sophora japonica (MMA) lectin (100 mg / ml, BoehringerMannheim) or Western elderberry (SNA) lectin (100 mg / ml, BoehringerMannheim) in the containing Incubate in MEM with 0.3% bovine serum albumin. After 48 hours of incubation, the medium was replaced ...
Embodiment 2
[0137] Materials and Methods
[0138] cell . 293T human embryonic kidney cells were cultured in Dulbecco's medium supplemented with 10% fetal calf serum (FCS), and Madindarby canine kidney (MDCK) cells were cultured in Eagle's medium supplemented with 5% newborn calf serum.
[0139] Plasmid-based reverse genetics . As described by Neumann et al. (1999), a plasmid containing the cNDA of the A / WSN / 33 (H1N1) viral gene under the control of the RNA polymerase I promoter and terminator (called the Pol1 plasmid) and pCAGGS / The MCS plasmid generates influenza A virus (Figure 4). Briefly, the Pol1 plasmid and protein expression plasmid were mixed with the infection reagent Trans ITLT-1 (Panvera, Madison, WI), incubated at room temperature for 10 minutes, and added to Opti-MEM (GIBCO / BRL) cultured 1×10 6 in 293T cells. 48 hours after transfection, 0.5 μg / ml trypsin was added to the medium to activate the HA protein, and incubated at 37°C for 1 hour. Collect the supernatant....
Embodiment 3
[0162] To obtain a realistic image of the viral content, electron microscopy tomography was performed (Fig. 11A). Images were taken of virus particles with a thickness of 50 nm. One of these virus particles is then analyzed to reconstruct a 3D image of the virus particle's contents. The structures (rods) within the particles were colored to distinguish each structure (Figures 1 IB-F, showing top, side and bottom views). Most views of the rod are in cross section, but in views where the rod is cut through the middle, the entire molecule is shown. However, all views show the interaction between the rods.
[0163] Based on the summary results above, and the data supporting that each viral fragment contains a unique sequence important for incorporation into the virion that may contribute to the unique morphological properties of the viral content, vRNA fragments are likely to select Sexually introduced into influenza virus particles. This information is useful not only for the...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Thickness | aaaaa | aaaaa |
| Titer | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More