红光熊蜂蜂毒蛋白酶的基因克隆和表达

Gene cloning and expression of Bombus ignitus venom protease from the Bumblebee,Bombus ignitus

目的克隆表达序列标签(ESTs)中筛选出的红光熊蜂蜂毒蛋白酶(BiVP)基因,在草地夜蛾(Spodoptera frugiperda)细胞Sf-9中进行表达。方法利用ESTs从红光熊蜂cDNA文库中获得BiVP基因,以PCR技术扩增出BiVP的完整cDNA,并构建病毒表达载体PBac1-BiVP,通过昆虫杆状病毒表达系统,在Sf-9昆虫细胞中进行表达,表达产物用快速蛋白液相色谱(FPLC)系统进行纯化。结果成功克隆BiVP基因并在Sf-9昆虫细胞中得到表达,表达产物纯化后经SDS-PAGE检查为单一条带。结论首次在红光熊蜂中克隆到蜂毒蛋白酶基因,在昆虫杆状病毒表达系统中进行了表达,并对表达产物进行了纯化,为深入研究BiVP的生物活性奠定了基础。

Objective To clone the Bombus ignitus venom protease （BiVP） gene screened from the expression sequence tags （ESTs） of the bumblebee, Bombus ignitus and express it in Spodoptera frugiperda Sf-9 cell. Methods The BiVP gene was screened by the ESTs technology and cloned by PCR method from the cDNA library of the bumblebee,Bombus ignitus. It was reconstructed to form the baculovirus transfervector pBacl-BiVP and expressed in Sf-9 insect cell. The recombinant BiVP was purified by the fast protein liquid chromatography system. Results The gene was cloned and expressed in Sf-9 insect cell. The recombinant protein purified showed a single band in SDS-PAGE. Conclusions It is the first time to clone the BiVP gene from the bumblebee, Bombus ignitus and express it in Spodoptera frugiperda Sf-9 cell. The purified recombinant protein fulfilled the needs of further studying the bioactivity of this protein.