柞蚕核型多角体病毒基因表达载体系统的构建与基因表达

Construction and Gene Expression of Antheraea pernyi Nucleopolyhedrovirus Gene Expression Vector System

柞蚕是一种主要分布在我国东北部山区、野外饲养的经济昆虫,以蛹滞育越冬,其蚕蛹具有个大、容易固定、保存时间长、无须饲养、容易运输等优点。利用柞蚕蛹作为生物反应器生产外来蛋白质,可以进行大规模机械化生产,并可减少操作上的烦琐和劳动力。以柞蚕核型多角体病毒(AnpeNPV)作为基因表达载体,在柞蚕培养细胞(AnPe细胞)和柞蚕蛹中成功地表达了β-半乳糖苷酶基因(LacZ),并利用AnPe细胞筛选、获得了纯化AnpeLacZ重组病毒。该重组病毒在TC-100(含10%FBS)培养的AnPe细胞中,感染后第12天β-半乳糖苷酶达到最高,酶活性为40.9units/ml,在SF-900Ⅱ培养的AnPe细胞中,感染后第18天β-半乳糖苷酶达到最高,酶活性为59.9units/ml,后者表达量稍高,但时间滞后。AnpeLacZ在5℃保存了7个月的柞蚕蛹中,感染后第15天酶活性达到最高,雌蛹为14.3units/g,雄蛹为11.7units/g,雌蛹比雄蛹略高。结果显示,柞蚕核型多角体病毒和柞蚕蛹可以作为一个可以机械化大规模生产的新型杆状病毒基因表达载体系统开发和利用。

Chinese oak silkworm, Antheraea pernyi is an economic insect rearing outdoors. It is mainly cultured in the area of Northeast of China. A. pernyi diapause by pupa stage for hibernation. The pupa is big, easy fix, keep time long, need not the feeding, easy conveyance etc. advantage. A. pernyi pupae as biological reactor product foreign protein may be large-scale mechanization production, reduce operational and trivial with labor force. A. pernyi nucleopolyhedrovirus （AnpeNPV） as gene expression vector successfully expressed β- galatosidase gene （LacZ） in A. pernyi culture cells （AnPe cell ） and diapause pupae, moreover purified recombinant AnpeNPV expressing LacZ （AnpoLacZ） obtained using AnPe cell. For β-galatosidase yield of the AnpoLacZ, their peak activity was 40.9 units/ml at 12 days post-infection （ p. i. ） in AnPe cell cultured using TC-100 medium （ 10 % FBS） and is 59.9 units/ml at 18 days p. i. in AnPe cell cultured using SF-900 Ⅱ medium, respectively. The latter was slightly high but slow at time. The β-galatosidase activity of AnpoLacZ in A. pernyi pupae who store 7 months at 5℃ reached peak at 15 days p. i. , female was 14.3 units/g and male was 11.7 units/g, respectively. The female was better than male. Results demonstrate that AnpeNPV and A. perny/ pupae could be developed and used as a novel baculovirus gene expression system which may be mechanization and large-scale production.