马铃薯Y病毒N株系HC-Pro基因在毕赤酵母中分泌表达与鉴定  

Secretion expression of the HC-Pro gene of Potato virus Y N strain in Pichia pastoris and identification of the protein

作　　者：杨栋[1] 吴云锋[1] 王睿[1] 

机构地区：[1]西北农林科技大学植物保护学院陕西省农业分子生物重点实验室,杨凌712100

出　　处：《植物病理学报》2007年第2期159-163,共5页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(30270876);教育部"创新团队建设计划"(No.200558)

摘　　要：以克隆载体pGEMT-PVY-NHC为模板,用PCR方法获得HC-Pro基因,构建表达载体pPIC9K-HC,将重组质粒经SalI单酶切后电转化PachiapichiaGS115菌株,筛选出对G418有高抗性和在MM培养基上生长缓慢的转化子。经摇瓶发酵和1%甲醇诱导后,SDS-PAGE检测发酵液上清,在66kD处有1个特异条带表达,目的条带蛋白占上清液总蛋白的40%。Westernblot鉴定表明,表达蛋白可以和HC抗体发生结合反应。Using plasmid pGEMT-PVY-NHC as a template, DNA of TFP11-161 was obtained by PCR and cloned into plasmid pPIC9K to construct expression plasmid pPIC9K-HC. After being linearized with restriction endonuclease Sal I, the recombinant plasmid was transformed into Pichia pastoris by electroporation. The transformants with high level of G418 resistance and slow growth on the MM plates were selected. After the selected transformants were grown in BMGY medium and induced by methanol, the culture supernatant was collected by centrifugation and analyzed by SDS-PAGE. The result showed that the recombinant HC-Pro molecular mass was about 66 kD and amounted to 40% total soluble proteins by gel analysis. Western blot analysis proved that the expression protein could bind to HC-Pro polyclonal antibody specifically.

关 键 词：HC-PRO基因 毕赤酵母 表达 

分 类 号：S432.41[农业科学—植物病理学] Q943.2[农业科学—农业昆虫与害虫防治]