Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors.
We have explored the possibility of improving baculovirus pesticides by incorporating an insect-specific neurotoxin gene into a baculovirus genome. A 112-bp gene (BeIt) encoding insectotoxin-1 of the scorpion Buthus eupeus was synthesized and cloned in Escherichia coli. For expression, BeIt was transferred to the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV). Three different recombinant AcMNPVs, carrying BeIt under the control of the strong AcMNPV polyhedrin promoter, were constructed and expression of BeIt was monitored upon infection of Spodoptera frugiperda (Sf) cells. Toxin expression was low using a recombinant virus in which BeIt was inserted 6 nucleotides (nt) downstream from the intact polyhedrin mRNA leader. More expression was observed when a signal-peptide was attached in-frame to the N terminus of BeIt. The highest level of expression was observed with a fusion gene comprised of the 58 N-terminal codons of polyhedrin fused to BeIt; however, the level of expression was ten- to twenty-fold below that for polyhedrin. Polyhedrin promoter-directed transcripts of all three recombinants accumulated to levels similar to those of wild-type polyhedrin transcripts, indicating that the limitation to expression of unfused BeIt was not at the level of transcription but rather at the posttranscriptional level including translation or protein stability. Paralytic activity of the toxin products was not detected.
Carbonell
L F
LF
Department of Genetics, University of Georgia, Athens 30602.
Hodge
M R
MR
Tomalski
M D
MD
Miller
L K
LK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005796:Genes; D005813:Genes, Synthetic; D005814:Genes, Viral; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012604:Scorpion Venoms / Q000235:genetics*; D012605:Scorpions / Q000235:genetics*
C047740:Buthus toxin I; D009498:Neurotoxins; D012604:Scorpion Venoms