Cloning and expression in Escherichia coli of the insecticidal delta-endotoxin gene of Bacillus thuringiensis var. israelensis

Abstract

Recombinant plasmids containing the mosquitocidal delta-endotoxin gene were constructed by inserting HindIII fragments of the Bacillus thuringiensis var. israelensis 72-75 Md plasmid in to the Escherichia coli vector pUC12. Two recombinants producing the 26 000 Da delta-endotoxin (pIP173 and pIP174) were identified by screening clones in an E. coli in vitro transcription-translation system. Both recombinants were 12.4 kb chimaeric plasmids comprising pUC12 and a common 9.7 kb HindIII fragment of the B. thuringiensis plasmid. The 26 000 Da polypeptide synthesis in vivo from pIP174 transformed into E. coli JM101 was lethal to mosquito larvae and cytotoxic to mosquito cells in vitro. The biological authenticity of the cloned product was further confirmed by demonstrating that the cytotoxicity of the polypeptide was neutralised by antiserum to the authentic delta-endotoxin or by preincubation with excess toxin receptor. Transcription of the recombinant delta-endotoxin gene in E. coli appears to utilise a Bacillus promoter sequence(s) rather than the pUC12 beta-galactosidase promotor.