失活 ChiA 和 v-cath 基因的重组BmNPV 表达 hGM-CSF

利用 DNA 同源重组技术使家蚕核型多角体病毒的多角体蛋白基因取代 v-cath 、 ChiA 两基因的部分编码区域和共同的启动子区域，获得了 v-cath 、 ChiA 两基因同时失活的、可形成多角体的、并能表达人粒细胞 - 巨噬细胞集落刺激因子的重组病毒 BmNPVpolh+CP－ChiA－GMCSF+. 研究结果显示： v-cath 、 ChiA 两基因的失活不影响病毒的复制及多角体的形成，但感染细胞的存活时间比野生病毒感染的多 2 天，可明显改善外源基因在培养细胞和家蚕中的表达水平；感染 BmNPV polh+CP－ChiA－GMCSF+的家蚕幼虫体表保持正常，无野生病毒感染后引起的破皮流脓现象 .

hGM-CSF Expression by Using Recombinant BmNPV With Inactivated Chitinase and Cathepsin Genes

Obtained by homologous recombination of replacing part coding region and common promotor region of cathepsin (v-cath) and chitinase (ChiA) genes with polyhedrin gene, the recombinant baculovirus BmNPVpolh+ CP-ChiA-GMCSF+, in which the v-cath and ChiA genes were inactivated, could express hGM-CSF gene and polyhedrin gene simultaneously. It was revealed that the inactivation of v-cath and ChiA genes had no significant effect on viral growth or polyhedron production in BmN cells, and the cells infected with the recombinant baculovirus BmNPVpolh+CP-ChiA-GMCSF+ survived 2 days more than those infected by BmNPV and BmNPV-hGMCSF, the expression level of foreign gene could be obviously improved. The B.mori larva infected with BmNPVpolh+CP-ChiA-GMCSF+ retained a healthy, normal white epidermis color, in contrast to the typical discoloration of the larval epidermis seen after death in wild-type BmNPV-infected larvae, whose cuticle becomes soft and easily punctured