国家高技术研究发展计划(863)资助项目(2004AA2Z1020).
This work was supported by a grant from Hi-tech Research and Development Program of China (2004AA2Z1020).
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 .
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.
贡成良,薛仁宇,曹广力,石晓燕,沈卫德.失活 ChiA 和 v-cath 基因的重组BmNPV 表达 hGM-CSF[J].生物化学与生物物理进展,2005,32(10):947-952
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