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The mosquito-larvicidal binary toxin produced by Bacillus sphaericus consists of two polypeptides: BinA and BinB. Both proteins function together, and maximum toxicity is obtained when both
are present in equimolar ratio. Cloning and expression of each component separately in heterologous hosts led to low toxicity
of the crystal proteins. To improve the expression level, the purification process, and the activity of the binary toxin,
the binA and binB genes were separately cloned in Eschericia coli. Each gene was fused in frame to the glutathione S-transferase (GST) gene to be expressed as GST-fusion protein (GST-BinA
and GST-BinB). A high expression level was observed from both constructs, and the fusion proteins exhibited high toxicity
to Culex quinquefasciatus larvae. High-purity toxin could be obtained by affinity chromatography. The result suggests that GST moiety facilitates high
protein production and enables better solubility of the toxin inclusions inside the larval gut, leading to higher toxicity
of the fusion protein. 相似文献
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Promdonkoy B Chewawiwat N Tanapongpipat S Luxananil P Panyim S 《Current microbiology》2003,46(2):94-98
The cytolytic delta-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 m M Na(2)CO(3), pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 microgram/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC(50) 0.5-1.0 microgram/ml). 相似文献
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Promdonkoy B Promdonkoy P Audtho M Tanapongpipat S Chewawiwat N Luxananil P Panyim S 《Current microbiology》2003,47(5):383-387
The binary toxin gene encoding BinA (42 kDa) and BinB (51 kDa) from Bacillus sphaericus strain 2297 was cloned and expressed in E. coli. Low expression level was found when both proteins were expressed from a single operon. High expression was observed when the gene encoding an individual protein was placed downstream of the T7 promoter. The expression level of BinB was not different when expressed alone (non-fusion) or as a fusion form with T7 peptide (T7-BinB). Both forms of BinB were equally stable. Unlike BinB, the non-fusion form of BinA was less stable than T7-BinA. The mosquito larvicidal test showed that BinA or BinB alone was not toxic to mosquito larvae, but high toxicity was found when both BinA and BinB were applied. The results suggest that a short peptide of T7 linked to the N-terminus of either BinA or BinB does not affect their toxicity, but may make the toxin, especially BinA, more stable. 相似文献
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Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4::Omega). We now report the successful transfer of two mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4::Omega, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the Omega region yielding two integrants designated An11S4::cry4B and An11S4::Omega::bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4::cry4B, but no expression of the binary toxin gene could be detected from An11S4::Omega::bin. Nevertheless, these two integrants exhibited mosquito-larvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels. 相似文献
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Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality of cultured Penaeus monodon in Asia. In this study, we investigated whether mouse polyclonal antiserum raised against the YHV gp116 or gp64 structural glycoproteins could neutralize YHV infectivity as determined using an in vitro quantal assay in primary cultures of lymphoid organ cells. Anti-gp116 antiserum showed virus-neutralizing activity whereas anti-gp64 antiserum failed to inhibit infection. The results suggest that gpl16 antiserum blocks binding of virions to cellular receptors to facilitate YHV entry into lymphoid organ cells. 相似文献
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