Optimization of the production frequency of recombinant AgMNPV

CHRISTINA B. MCCARTHY; ELOÍSA I. ARANA; ALICIA SCIOCCO-CAP; VICTOR ROMANOWSKI

Noordwijkerhout

Congreso; 34th Annual Meeting of the Society for Invertebrate Pathology; 2001

Society for Invertebrate Pathology (SIP)

Anticarsia gemmatalis nucleopolyhedrovirus (AgMMV) is the most widely used baculovirus pesticide. However, its low speed of action limits its use. Our laboratory is developing a project which includes the generation of more efficient AgMNPV (rAgMNPV). The low proportion of recombinants (1% or less, the majority of which are single cross recombinants) obtained by homologous recombination after cotransfection of viral and plasmid DNAs, can be improved using linearized viral DNA. To this end we designed a strategy for the insertion of unique restriction sites in the parental AgMNPV genome. The first step consisted in introducing I-PpoI (intron encoded endonuclease) restriction sites in a plasmid that contained the E. coli LacZ gene flanked by the upstream and downstream non-coding regions of AgMNPV polyhedrin gene (pAgPhZ3). The cotransfection of this plasmid and AgMNPV DNA generated a IacZ+ and polh- rAgMNPV with unique restriction sites. A control plasmid and the linearized AgMNPV DNA were used to cotransfect cells to evalua the increase in the yield of recombinants. This virus will be used as parental for the production of a series of recombinants.