苜蓿银纹夜蛾核多角体病毒在家蚕蛹体内的复制及重组病毒外源基因的表达

家蚕蛹在复眼着色期,经4℃冷藏24小时后,可被Ac NPV(苜蓿银纹夜蛾核多角体病毒)感染。在蛹体内复制出的多角体大小差异较大,且比在昆虫Sf—21细胞中繁殖的Ac NPV和在蚕蛹体内形成的Bm NPV多角体小。在蛹体内繁殖的Ac NPV的游离病毒可以回返感染Sf—21细胞。由蚕蛹内分离的Ac NPV基因组DNA的限制性内切酶图谱与野生型的Ac NPV相同。以上结果证明Ac NPV可以在蚕蛹体内复制。含HBsAg基因(人乙肝表面抗原)的重组Ac NPV亦可感染家蚕蛹,并能正确表达外源基因。此外,还发现化蛹后第2天的家蚕蛹被注射约5×10~5PFU的Ac NPV,可诱导“人工滞育蛹”现象的发生,在25℃经过30天后蛹仍存活,发育停滞在复眼着色前阶段。

REPLICATION OF Ac NPV AND FOREIGN GENE EXPRESSION OF RECOMBINANT Ac NPV IN SILKWORM PUPA

After the silkworm pupa at the compound eye darkened stage was stored in 4℃ for 24h, it could be infected by Ac NPV (Autographa californica Nuclear polyhedrosis Virus). The sizes of Ac NPV polyhedra replicated in the silkworm pupa are very different. And they are smaller than those of Ac NPV polyhedra replicated in Sf-21 cell line and those of Bm NPV polyhedra replicated in the silkworm pupa. The extracellular budded virus of Ac NPV reproduced in the silkworm pupa infected Sf -21 cell line again. The viral genome DNA of Ac NPV separated from the silkworm pupa showed the restriction enzyme map,which was identical with that of the wild type Ac NPV. All above results demonstrated Ac NPV could be reproduced in the silkworm pupa. The recombi-nant Ac NPV containing HBsAg(Human Hepatitis B Virus Surface Antigen S) gene was also expressed in the silkworm pupa. In addition,it was discovered that the silkworm pupa in the early development stage could be induced to "artificial diapause pupa "after it was injected about 5×105 PFU Ac NPV. The pupa lived over 30 days in 25℃. But the development of pupa stopped at the stage of the pre-compound eye darkened.