This study describes the generation of cold-adapted recombinant influenza viruses using bi-directional plasmids. The methodology involves transfecting cultured human epithelial-like cells with plasmids encoding viral proteins, leading to the production of live-attenuated viruses suitable for vaccine preparation.
Begin with bi-directional plasmids carrying cDNA encoding certain viral essential proteins of a cold-adapted influenza strain.
Introduce a new set of bi-directional plasmids containing hemagglutinin and modified neuraminidase cDNA from a different influenza strain.
Introduce a transfection reagent to this mix and incubate to form a complex with the plasmids.
Transfer these complexes onto cultured human epithelial-like cells to enable plasmid entry into the cell.
The bi-directional plasmids with two promoters in opposite orientations enable the production of viral proteins and their corresponding RNA.
Incubate the cells at a lower temperature.
The viral proteins and the viral RNAs self-assemble to form cold-adapted recombinant influenza viruses with reduced replication in warmer conditions.
Overlay with sialic acid-overexpressing MDCK cells and modified trypsin enzymes that allow viral attachment and entry inside cells.
Incubate at the same temperature to induce replication and production of new viruses.
Centrifuge and harvest the recombinant live-attenuated influenza viruses ready for a vaccine preparation.
To begin, plate a million 293T cells in each well of a 6-well plate with DMEM containing 10% fetal bovine serum and 1% pen strep. Next, prepare enough plasmid transfection mixture for 100 microliters per well. Combine a microgram of each plasmid and fill the tube with pre-warmed Opti-MEM cell medium to 50 microliters. In another tube, add twice the volume of lipofectamine 2000 corresponding to the total quantity of plasmid to be transfected, and top the tube off to 50 microliters with Opti-MEM.
Before adding the transfection mix to the cells, let it sit for 30 minutes at room temperature. Then, add 100 microliters of the transfection mix to each well and move the plate to an incubator for 16 hours. The next day, change the medium to DMEM with 0.3% BSA and 1% pen strep. Then. move the cells to 33 degrees Celsius with 5% carbon dioxide for five hours.
After five hours, add an overlay of a million influenza-permissive MDCK cells in trypsin-supplemented DMEM. Maintain the co-cultures for 2 to 3 days under the 33 degrees Celsius incubation regime to generate and amplify the virus. Later, clear out the debris using a five-minute 260 g spin, then collect the supernatants.
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