棉铃虫组织蛋白酶B在杆状病毒表达系统中的表达及鉴定

以棉铃虫（Helicoverpa armigera）组织蛋白酶B作外源基因重组构建克隆载体，自重组菌株HCB-DH10Bac、Histag-HCB-DH10Bac中提取穿梭质粒，脂质体法转染草地贪夜蛾卵巢细胞系Sf21细胞，转染液再感染细胞，收集感染3～4d的上清液，提取芽生病毒的DNA，PCR法鉴定外源HCB基因，感染上清进行SDS-PAGE、Western-blotting、蛋白酶活性检测．结果：感染上清中的芽生病毒的DNA作模板扩增出预期的1700bp片段，SDS-PAGE、westem-blotcing检测均在28ku处有明显表达产物，且表达产物有蛋白水解活性．

Expression and identification of Helicoverpa armigera cathepsin B in baculovirus system

Recombinant cloning vectors with Helicoverpa armigera cathepsin B gene were constructed. After being isolated , the recombinant bacmids were used for transfection into Sf21 cells with lipofectin. The supernatant of transfection was used for infection into Si21 cells. From the supernatant, budded virus was extracted in order to identify HCB gene by PCR. The supernatant was analyzed by assays of SDS-PAGE, westem-blotting and protease activity analysis. The result of PCR shows that DNA fragment of 1 700 bp has been obtained which is correlated to the length of HCB gene and the vector ann. A new protein with MW 28 ku has been found in the supematant by SDS-PAGE and western-blotting and it also shows proteolysis activity in the experiment of pmtease activity.