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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 92, No. 2, 1997, pp. 257-262
Bioline Code: oc97051
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 92, No. 2, 1997, pp. 257-262

 en Cloning, expression and toxicity of a mosquitocidal toxin gene of Bacillus thuringiensis subsp. medellin
Nora Restrepo; Diana Gutierrez; Maria M Patino; Isabelle Thiery; Armelle Delecluse & Sergio Orduz

Abstract

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal
crystalline inclusions which are toxic to mosquito larvae. It has been
shown that the inclusions of this bacterium contain mainly proteins of 94,
68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by
using the Lambda Zap II cloning kit. Recombinant plaques were screened with
a mouse policlonal antibody raised against the 94 kDa crystal protein of
Btmed. One of the positive plaques was selected, and by in vivo excision, a
recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa
toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then
subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing
the recombinant plasmid pBTM3 and used to transform by electroporation Bt
subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to
mosquito larvae was estimated by using first instar laboratory reared
Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole
crystals. Toxicity results indicate that the purified inclusions from the
recombinant Bti strain were toxic to all mosquito species tested, although
the toxicity was not as high as the one produced by the crystal of the
Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that
the inclusions produced by the recombinant strain Bti (pBTM3) were mainly
composed of the 94 kDa protein of Btmed, as it was determined by Western
blot.

Keywords
Bacillus thuringiensis - crystal protein - gene cloning and expression - recombinant - mosquito larval toxicity

 
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