A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus

Abstract

Abstract. The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT 50 and LD 50 bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.* Address correspondence to Xiao-Guang Chen, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. E-mail: xgchen@fimmu.com † These authors contributed equally to this work. THE PATHOGENICITY OF RECOMBINANT AEDNA ON AEDES ALBOPICTUS1 protein (NS1), and structural protein (VP) genes were transcribed from the P7 and P61 promoters in the genome, respectively, which has been described in detail previously. 15The p7NS1-GFP expresses an NS1-GFP fusion protein from the p7 promoter. The construction of p7NS1-GFP is described in detail elsewhere. 15The p7NS1-BIT-GFP was derived from p7NS1-GFP, and BmK IT1 coding sequences were inserted at the AgeI site of p7NS1-GFP. As a result, the BmK IT1-GFP construct was fused to the C-terminus of NS1. An additional flexible linker (GGGGS) was used to link these two fragments. 16 The NS1-linker-BmK IT1-GFP fusion protein was expressed from the p7 promoter.The p61NTS-BIT-GFP was derived from pUCA. The BmK IT1-GFP coding sequence was cloned into the structural protein, ORF (2,791-3,635 nt). As a result, the 77 aa of the N-terminal of the structural protein, VP1, which harbors a putative nuclear targeting signal (NTS) sequence, 17 were fused to the N-terminus of the BmK IT1-GFP protein, and the fusion protein was expressed from the p61 promoter.The p7NS1-BIT was derived from p7NS1-BIT-GFP by deleting GFP from C-terminus of BmK IT1. As a result, the NS1-linker-BmK IT1 fusion protein was expressed from the p7 promoter.All of the constructions were confirmed by sequencing (data not shown). The plasmids that were used in this study are depicted in Figure 1 .Mosquito cell maintenance and transfection. Aedes albopictus C6/36 cells (ATCC CRL-1660) were grown at 28°C in RPMI 1640 medium (Gibco BRL, USA ) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) and 1% antibiotics (Penicillin-Streptomycin; Gibco BRL). The C6/36 cells were grown to a 50-70% confluent monolayer in 24-well cell culture plates (Corning costar, USA ) and washed onc...

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