Objective: To assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects.
Methods: The AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested.
Results: AaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LC50 of 3.00×106 cells/mL and 1.25×107 cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002).
Conclusion: The B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.
目的: 构建表达蝎毒素AaIT或苏云金杆菌毒素Cyt2Ba的大肠埃希菌，检测重组菌对致倦库蚊和白纹伊蚊II龄幼虫的毒力，并测试不同剂型的增效作用。
方法: 分别将AaIT和Cyt2Ba编码基因克隆于pET28a（+）质粒上，并将重组质粒分别转化BL21 E.coli获得分别表达AaIT和Cyt2Ba的工程菌株，经IPTG诱导后，用SDS-PAGE和Western-blot检测重组蛋白的表达；测试不同浓度工程菌菌液对于蚊幼虫的杀灭效果；并将有效的工程菌制成干粉剂，检测其干粉末及其配剂对蚊幼虫的杀灭效果。
结果: AaIT和Cyt2Ba重组蛋白在大肠埃希菌中成功表达，但AaIT重组蛋白对蚊幼虫不具有毒性。表达Cyt2Ba重组蛋白的工程菌菌液对白纹伊蚊和致倦库蚊幼虫均具有明显的杀灭作用，48 h的LC50分别为3.00×106和1.25×107 cells/mL，其中对白纹伊蚊幼虫的杀灭效果要显著优于对致倦库蚊幼虫的杀灭效果（P < 0.001），并且表达Cyt2Ba重组蛋白的工程菌干粉末及其配剂均具有较好的杀蚊幼虫效果。当该工程菌干粉末与干酵母粉、小麦粉和白胡椒粉分别按4:1、1:1和1:4比例制成配剂处理蚊幼虫48 h后，结果显示当质量比为1：1时，配剂杀蚊幼效果显著提高（P=0.044），并且白胡椒粉末配剂效果要显著好于干酵母粉和小麦粉配剂（P=0.002）。
结论: 表达Cyt2Ba的重组大肠埃希菌对蚊幼虫具有较好的杀灭作用，合适配剂的使用可以增强其在蚊虫防制中的效果。