球形芽胞杆菌Cry48Aa/Cry49Aa杀蚊毒素与致倦库蚊中肠上皮细胞复合物的结合特性

The Binding Kinetics of Lysinibacillus sphaericus Cry48Aa/Cry49Aa Toxin to Brush-border Membrane Fractions of Culex quinquefasciatus Larvae

Cry48Aa/Cry49Aa毒素是近年来在球形芽胞杆菌IAB59中发现的新型双组分毒素,仅对库蚊具有较高的毒杀作用并能杀死二元毒素(Bin)抗性库蚊,是一种比较有潜力的新型杀蚊毒素蛋白,但目前对Cry48Aa/Cry49Aa毒素的作用模式还不清楚。本研究将cry48Aa和cry49Aa基因在苏云金芽胞杆菌无晶体突变株中表达,纯化毒素的生测结果表明该复合毒素对Bin毒素敏感和抗性致倦库蚊均表现较高的毒杀作用,毒力无显著差异。生物素标记的毒素与致倦库蚊BBMF特异性结合试验表明Cry48Aa和Cry49Aa毒素与两蚊虫品系BBMF都具有较高的结合特异性,Cry48Aa结合能力较高,其解离常数(Kd)分别为(9.5±1.8)和(13.9±2.3)nmol/L;Cry49Aa的解离常数分别为(25.4±3.8)和(28.1±4.2)nmol/L。异源竞争结合试验结果表明Cry48Aa毒素则可以有效地和Cry49Aa毒素竞争结合BBMF蛋白上的结合位点,其IC50分别为(22.1±3.7)和(15.4±2.6)nmol/L,而Cry49Aa不能竞争封闭Cry48Aa毒素与两蚊虫品系BBMF蛋白的结合位点,其IC50均大于17μmol/L。该研究结果可为揭示Cry48Aa/Cry49Aa毒素的作用机制提供一定的研究基础。

The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Lysinibacillus sphaericus strain IAB59, with high toxicity against binary toxin （Bin） susceptible and resistant Culex spp. However, its action mode against the target mosquitoes has not been elucidated. In this work, the cry48Aa and cry49Aa genes were individually cloned and expressed in the crystal minus B. thuringiensis. The purified Cry48Aa/Cry49Aa toxin showed similar high toxicity against Bin-susceptible and resistant C. quinqueJ＆sciatus. Direct-binding assays and competition assays were used to identify specific binding between the biotinylated toxin and brush-border membrane fractions （BBMF） of the susceptible and resistant C. quinquefasciatus larvae. The results showed that Cry48Aa had a higher binding affinity, with a dissociation constant （Kd） of （9.5 ± 1.8） nmol/L and （13.9±2.3） nmol/L, compared to the Kd of （25.4±3.8） and （28.1 ±4.2） nmol/L for Cry49Aa. In heterologous competition binding assays, Cry48Aa toxin could effectively compete with Cry49Aa toxin for binding sites on BBMF from both mosquito larvae, with ICs0 of （22.1 ±3.7） nmol/L and （15.4±2.6） nmol/L, respectively, whereasCry49Aa （with IC50〉 17 gmol/L） could not compete and block the binding sites of Cry48Aa toxin. These results provide important information for the preliminary action mechanism of Cry48Aa and Cry49Aa.