[Establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus BAC-TO-BAC expression system]

Present studies describe the successful establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) BAC-TO-BAC expression system. The mini-F-lacZ-attTn7-kan fragment (Luckow et al, 1993) was inserted into SeMNPV US1 isolate (SeUS1) at polyhedrin gene locus by directly cloning. The recombinant virus containing low-copy-number mini-F replication, which named bacmid, can propagate in Escherichia coli. Because SeUS1 isolate is make up of several genotypes and one bacmid carries one SeMNPV genotype, the SeUS1 BAC library is established by all SeMNPV bacmids (SeBAC). REN analysis for 111 SeBAC shows that SeUS1 consists of the genotype with whole SeMNPV genetic information and several genotypes with various different deletions. Progeny virus can be produced in insect cell line after transfection with SeBAC10, which carries the whole SeMNPV genome. So SeBAC10 is a shuttle vector that can replicate in eukaryocyte as well as prokaryocyte. Considering the insert mutation of SeMNPV polyhedrin gene (Seph) in SeBAC10, Seph was reintroduced into the bacmid by site-specific transposon-mediated insertion at attTn7, the target site for the bacterial transposon Tn7. The derived recombinant SeBAC10 was named SeBAC10ph. After SeBAC10ph was transfected into Se301 cells (a susceptible insect cell line to SeMNPV), cytopathogenic effect was shown and polyhedra appeared, which indicate that the foreign gene (Seph) is expressed.