Establishment of NewTrichoplusia niCell Lines in Serum-Free Medium for Baculovirus and Recombinant Protein Production

Abstract

Thirty one new cell lines fromTrichoplusia nieggs have been established in Ex-Cell 400, a commercially available serum-free medium. These cell lines were evaluated for support of baculovirus replication with the 1A strain ofAutographa californicaMNPV (AcMNPV-1A) and withT. niSNPV (TnSNPV). All cell lines were found to be susceptible to both viruses. Those cell lines demonstrating superior qualities were chosen for further study. Of the new cell lines tested, the BTI-Tn-4B31 (Tn-4B31) cell line produced more AcMNPV and TnSNPV budded virus (8.8×107PFU/ml and 5.7×106PFU/ml, respectively). This cell line also produced the greatest number of AcMNPV occlusion bodies (OBs) per cell (104), when compared to the other new cell lines. The BTI-Tn-4A14 (Tn-4A14) cell line produced the greatest number of TnSNPV OBs per cell (968) when compared to the other new cell lines. However, the highest producer of OBs/cell for both AcMNPV and TnSNPV was the BTI-Tn-5B1-4 (Tn-5B1-4 or High Five) cell line (127 and 1,152, respectively). Bioassay results with AcMNPV-1A showed that OBs derivedin vitrofrom all new cell lines were infectious toT. nineonate larvae when compared to larval-derived OBs. Expression levels of two recombinant AcMNPV viruses, E2-β-Gal and AcSEAP, were compared to IPLB-Sf-21 (Sf-21) cells and to Tn-5B1-4 cells, which are known to express recombinant proteins at high levels. Under identical conditions, β-galactosidase and alkaline phosphatase production in at least two cell lines, BTI-Tn-4B (Tn-4B) and Tn-4B31, were similar to Tn-5B1-4 cells and higher than Sf-21 cells. Preliminary characterization of different cell lines through random amplified polymorphic DNA analysis revealed unique DNA banding profiles for Tn-5B1-4 and Sf-21 cell lines, which distinguished them from each other and the Tn-4A14, Tn-4B, and Tn-4B31 cell lines. Similar DNA banding profiles were obtained for the Tn-4A14, Tn-4B, and Tn-4B31 cell lines, using multiple 10-mer primers.