[Construction and bioassay of secondary recombinant baculovirus with two foreign gene]

Plasmid pAcLEneo which bears neomycin gene derived by the baculovirus IE1 promotor was digested and the gene was harvested. A transfer vector pAc34DZ2 in which the polyhedrin envelope gene has been inactivated by insertion of expression cassette of PIE1/neo was constructed by inserting the neo cassette into the SacI site of plasmid pAc34DZ1. We have constructed the polyhedrin positive recombinant virus (I) vAcPhBtT which was able to express Bt truncated endotoxin gene. In order to improve the insecticide efficiency of recombinant virus (I), an anti-neomycin recombinant virus (II) vAcPhBtTPE- was obtained by second co-transfection into the Sf9 cells with pAc34DZ2 and recombinant virus (I) DNA. Southern blot and SDS-PAGE analysis indicated that the recombinant virus (II) was still able to express the 80 kD Bt truncated delta-endotoxin, but did not express the 34 kD polyhedrin envelope protein. The recombinant virus (II) without envelope released virion faster than recombinant virus (I) after alkaline lysis. Bioassay was carried out using Spodoera exigua. LC50 of the recombinant virus (II) was about half of wild type virus and LT50 reduced about two days.