Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin.
The delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki HD-73 was inserted into Autographa californica nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the delta-endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a polyhedrin-positive virus. Analysis of infected cell extracts showed that the delta-endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to Trichoplusia ni larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the B. thuringiensis delta-endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD50 value. The polyhedrin-positive virus had an LD50 value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.
Merryweather
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AT
Institute of Virology and Environmental Microbiology, Oxford, U.K.
Weyer
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U
Harris
M P
MP
Hirst
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M
Booth
T
T
Possee
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RD
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001483:Base Sequence; D004731:Endotoxins / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006460:Hemolysin Proteins; D015151:Immunoblotting; D007304:Insect Viruses / Q000235:genetics* / Q000302:isolation & purification; D007313:Insecta; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000077183:Occlusion Body Matrix Proteins; D015345:Oligonucleotide Probes; D010572:Pest Control, Biological; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D000077183:Occlusion Body Matrix Proteins; D015345:Oligonucleotide Probes; D014764:Viral Proteins; D015678:Viral Structural Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C009829:polyhedrin protein, Nucleopolyhedrovirus