中文摘要：

基于家蚕微孢子虫（Nosema bombycis,Nb）基因组中发现的蓖麻毒素B链（ricin B chain）序列数据设计引物,提取感染Nb第8天的家蚕幼虫中肠组织总RNA,利用RT-PCR方法扩增出RBL463-4基因,并将其克隆进毕赤酵母表达载体pPICZα-A中,构建重组酵母表达载体pPIC-R4。重组质粒pPIC-R4经SacⅠ线性化后,电击导入毕赤酵母X33中,利用博莱霉素（zeocin）筛选获得重组酵母X33/RBL463-4。以2%甲醇诱导表达后,经SDS-PAGE、Western blot检测表达产物,结果出现25kD和20kD2种蛋白,其中25kD蛋白与预测融合蛋白大小一致,表明获得了RBL463-4的融合蛋白。研究结果为进一步探究RBL463-4蛋白在家蚕微孢子虫侵染宿主过程中的功能作用奠定了基础。

英文摘要：

Based on the ricin B chain sequence data of Nosema bombycis,a pair of RT-PCR primers was designed to amplify cDNA of RBL463-4 gene from total RNA extracted from midgut of Bombyx mori larvae at day 8 post infection by N.bombycis.The obtained cDNA was subcloned into Pichia pastoris expression plasmid pPICZα-A to yield the recombinant plasmid pPIC-R4,which was subsequently linearized by restriction enzyme Sac I and transformed into P.pastoris X33 through electroporation.The recombinant yeast X33/RBL463-4 was obtained through screening with zeocin.After induction with 2% methanol,two proteins （25 kD and 20 kD） were detected in the fermentation supernatant by SDS-PAGE and Western blot.Size of the 25 kD protein was close to that of the predicted fusion protein,meaning that the fusion protein of RBL463-4 had been obtained.The above results established a good basis for further exploration on functions of RBL463-4 protein in the infection process of N.bombycis.