拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体，与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN，经200μg/ml zeocin抗生素筛选一个月，成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒，其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染，结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主，却并不在宿主体内形成包涵体，从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题，通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。

Potential Industrial Production of Foreign Protein in Silkworm Using the Collusion Bodies of Recombinant Bombyx mori(silkworm) Multiple Nucleopolyhedrovirus Rescuesd by Polyhedrin Expressing Stable Cell Line

The transposon donor vector harboring both polyhedrin gene droved by BmA3 promoter and zeocin resistant gene controlled by IE1 promoter from OpNPV co-transefeced with a lepidoptera transposition helper plasmid pie2piggBac into BmN cells. A polyhedrin expressing stable cell line was constructed by screening with the 200μg/ml zeocin selection medium for one month. Occlusion bodies (OB) were rescued successfully in the polyhedrin expressing BmN cells infected with recombinant BmBac-gfp budded virus without polyhedrin gene. The amount of rescued OB produced in polyhedrin expressing cells is only 8% of wild type BmNPV OB produced in normal cells. The silkworm larvae were infected efficiently when they were feed by the rescued OB sprayed mulberry leaves. Foreign GFP was expressed efficiently and no OB was observed in the hemolymph of silkworm larvae. This rescued OB oral infection method is benefit to promote the industrial progress of silkworm bioreactor by eliminating the tedious injection budded virus solution by hand.