ESTABLISHMENT OF NEW TRICHOPLUSIA NI CELL-LINES IN SERUM-FREE MEDIUM FOR BACULOVIRUS AND RECOMBINANT PROTEIN-PRODUCTION

Thirty one new cell lines from Trichoplusia ni eggs have been establis hed in Ex-Cell 400, a commercially available serum-free medium. These cell lines were evaluated for support of baculovirus replication with the 1A strain of Autographa californica MNPV (Ac-MNPV-1A) and with T. ni SNPV (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 x 10 (7) PFU/ml and 5.7 x 10(6) PFU/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 p roducer of OBs/cell for both AcMNPV and TnSNPV was the BTI-Tn-5B1-4 (T n-5B1-4 or High Five) cell line (127 and 1,152, respectively). Bioassa y results with AcMNPV-1A showed that OBs derived in vitro from all new cell lines were infectious to T. ni neonate larvae when compared to l arval-derived OBs. Expression levels of two recombinant AcMNPV viruses , E2-beta-Gal and AcSEAP, were compared to IPLB-Sf-S1 (Sf-21) cells an d to Tn-5B1-4 cells, which are known to express recombinant proteins a t high levels. Under identical conditions, P-galactosidase and alkalin e 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 cel ls. Preliminary characterization of different cell lines through rando m amplified polymorphic DNA analysis revealed unique DNA banding profi les for Tn-5B1-4 and Sf-21 cell lines, which distinguished them from e ach 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 cel l lines, using multiple 10-mer primers. (C) 1998 Academic Press.