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

柞蚕是一种野外饲养的经济昆虫，主要分布在我国东北部山区。柞蚕以蛹滞育越冬，其蚕蛹个大、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，可以大规模机械化生产，减少操作上的烦琐和劳动力。本文利用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、纯化获得了AnpeLacZ重组病毒。该重组病毒的β-半乳糖苷酶产量，在TC-100(含10%FBS)培养的AnPe细胞中最高酶活性为感染后第12天的40.9 units/ml，在SF-900Ⅱ培养的AnPe细胞中最高酶活性为感染后第18天的59.9 units/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14.3 units/g，雄蛹11.7 units/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。

Construction and Gene Expression of Antherae Pernyi Nucleopolyhedrovirus Gene Expression Vector System

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. In this paper, 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 (AnpeLacZ) obtained using AnPe cell. For β–galatosidase yield of the AnpeLacZ, 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-900Ⅱ medium, respectively. The latter was slightly high but slow at time. The β–galatosidase activity of AnpeLacZ 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. pernyi pupae could be developed and used as a novel baculovirus gene expression system which may be mechanization and large-scale production.