【摘要】 以重组质粒pGEM-3-1E为模板,扩增了序列两端分别含有EcoRⅠ和XbaⅠ酶切位点的堆形艾美球虫(Eimeria acervulina)广东株3-1E基因(长度为529 bp),将3-1E基因克隆至巴斯德毕赤酵母分泌型表达载体pPICZαC中,构建了酵母表达质粒pPICZαC-3-1E。转化毕赤酵母X-33得到含有3-1E基因的重组酵母,甲醇诱导产生的目的蛋白经SDS-PAGE分析和免疫印迹检测,表明毕赤酵母成功表达了3-1E基因。更多还原

【Abstract】 A pair of primers was re-designed on the basis of 3-1E gene sequence of Eimeria acervulina strain from Guangdong,and with the recombinant plasmid pGEM-3-1E as template,the 3-1E gene appro-(ximately) 529bp in size was amplified.After the 3-1E gene was cloned into plasmid pPICZαC with EcoRⅠ and Xba Ⅰ sites,a recombinant expression plasmid pPICZαC-3-1E was constructed successfully.After li-(nearization) with SacⅠ enzyme,the recombinant plasmid was transformed into wild type X-33 Pichia pastoris.Expression of positive yeast transformants was induced with methanol,resulting in production of a secretory fusion protein of approximately 27ku in size.Western blotting analysis showed that positive sera from recombinant protein-immunized mice evinced specific immunoreactivity with antigens of E.acervulina sporozoites.更多还原