Tnm fh insect medium

Example 7

The Spodoptera frugiperda Sf21 or SP cell lines were cultured in 6-well tissue culture plates (1×10⁶ cells/well) in TNM-FH insect medium (Pan Biotech™, Germany) containing 10% heat-inactivated fetal bovine serum (Pan Biotech™, Germany) at 27° C. AcMNPV recombinant baculoviruses were obtained by the “Bac-to-Bac” Baculovirus Expression System (Invitrogen™, USA). The different TB(+) transfer vectors containing the recombinant DNA regulatory elements were generated using the pFastBac™-DUAL plasmid (Invitrogen™). These transfer vectors were used to transfect Sf21 cells with Cellfectin® (Invitrogen™, USA). The resulting recombinant baculoviruses from the infection of Sf21 cells were then passaged twice in cells and titered by the plaque assay method. The obtained gene constructs of the TB (+) baculovirus expression cassettes are schematically shown in FIGS. 4, 7 and 10, showing the combinations of genetic regulatory elements involved in the genes expression (polhAc-ie-01/hr1p6.9p10, SEQ ID NO.: 17, plus the sequence of the gene coding for the desired protein, e.g., SEQ ID NOs: 26, 30, 32 and 33). The different expression cassettes were used to generate the recombinant baculoviruses used in the examples shown in FIGS. 6, 8, 9, 11, 12, 13, and 14.

Example 7

The Spodoptera frugiperda Sf21 or Sf9 cell lines were cultured in 6-well tissue culture plates (1×10⁶ cells/well) in TNM-FH insect medium (Pan Biotech™, Germany) containing 10% heat-inactivated fetal bovine serum (Pan Biotech™ Germany) at 27° C.

Confluent Sf9 or Sf21 cells in monolayer (1×10⁶ cells/well) were infected with the baculoviruses at different multiplicities of infection (from 0.01 to 10). In suspension, Sf9 cells (2×10⁶ cells/ml) were infected equally at different multiplicities of infection. Infected cells were analysed from 16 to 120 h post-infection.