携带苏云金芽胞杆菌cry1Ac10基因重组杆状病毒的构建及其杀虫活性

用Bac-to-Bac系统，构建了包含极晚期基因ph启动子驱动的带有全长苏云金芽胞杆菌cry1Ac10基因和完整多角体基因的重组质粒pFCP,用该重组质粒感染昆虫Sf9细胞，得到了带有多角体和能够表达cry1Ac10基因的重组杆状病毒vFcph，并在昆虫细胞中表达了Cry1Ac10蛋白。同时构建了含cry1Ac10的穿梭载体．pHTC，并分别转化大肠杆菌、枯草杆菌和苏云金杆菌晶体缺陷型菌株，结果表明此三种工程菌均表达了分子量为133．3kDa的原毒素蛋白，其中在苏云金芽胞杆菌中的表达量最高。生物测定表明重组杆状病毒vFcph的表达产物具有杀虫活性，能增加杆状病毒力，加快杆状病毒杀虫速度，说明利用杆状病毒极晚期基因启动子驱动苏云金芽胞杆菌杀虫晶体蛋白表达，从而改善杆状病毒的杀虫特性是可行的。

Construction and Bioactivity of Recombinant Baculovirus with cry1Ac10 Gene of Bacillus thuringensis

Using Bac-to-Bac system, the transfer vector pFCP with full-length cry1Ac10 gene of Bacillus thuringensis drived by the very late expressed gene ph promoter and intact polyhedra gene was constructed successfully. The recombinant virus vFcph , with complete polyhedra and cry1Ac10 gene, was obtained by transfection of the transfer vector pFCP DNA into insect Sf9 cells and cry1Ac10 protein can be expressed in insect cells. Meanwhile, a new shutter vector pHTC with cry1Ac10 gene was constructed. Three kinds of engineered bacteria produced by transformation of Bacillus thuringensis, Escherichia coli and Bacillus subtilis all expressed protoxin with the molecular weight of 133.3kDa. The expression of the protoxin in Bacillus thuringensis was the highest. Bioassay indicated that the expressing product can increase virulence and killing speed of baculovirus. The results indicated that it is feasible to enhance the effectiveness of baculovirus by inserting an insecticide crystal protein gene into baculovirus controlled by the promoter of very late expressed gene.