【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。更多还原

【Abstract】 Using plasmid pGEMT-PVY-NHC as a template,DNA of TFPI1-161 was obtained by PCR and cloned into plasmid pPIC9K to construct expression plasmid pPIC9K-HC.After being linearized with restri-ction 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.更多还原