家蚕微孢子虫蓖麻毒蛋白B链基因RBL463-4的克隆及在毕赤酵母中的表达  

Cloning of Ricin B Chain Gene (RBL463-4) from Nosema bombycis and Expression in Pichia pastoris

作　　者：李能章[1,2] 潘国庆[1] 李田[1] 贾立丽[1] 邓远洪[1] 周泽扬[1,3] 

机构地区：[1]西南大学蚕学与系统生物学研究所,重庆400716 [2]西南大学动物科技学院重庆市牧草与草食家畜重点实验室,重庆400715 [3]重庆师范大学生命科学学院,重庆400047

出　　处：《蚕业科学》2010年第6期957-961,共5页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金重点项目(No.30930067);重庆市自然科学基金重大项目(No.CSTC2006AA5019);国家重点基础研究发展计划"973"项目(No.2005CB121003)

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。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.

关 键 词：家蚕微孢子虫 蓖麻毒素B链蛋白 RBL463-4基因 毕赤酵母表达 免疫印迹法 

分 类 号：S884.21[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]