禽流感病毒NA基因在巴斯德毕赤酵母系统中的表达

Expression of avian influenza virus NA gene of Pichia pastoris

采用PCR扩增禽流感病毒 (AIV)NA基因 ,克隆入 pMD18 T载体中 ,再亚克隆入含有AOX1启动子和α分泌信号肽序列的巴斯德毕赤酵母 (Pichiapastoris)表达载体pPIC9k中 ,构建了重组转移载体 pPIC9kNA。经电穿孔转化酵母宿主菌GS115和筛选高拷贝重组转化子及筛选His+Mut+表型转化子后 ,摇瓶培养 ,10mL/L甲醇诱导表达后 ,经SDS PAGE、Western blotting、双向琼脂扩散试验、神经氨酸酶试验分析证明 ,获得了几株高效表达重组表达株 ,并且该重组NA蛋白具有免疫学活性。

NA gene of avian influenza virus (AIV) was amplified by PCR and then was cloned into the plasmid vector pMD 18-T .The complete coding region of the NA gene of AIV was then subcloned into the Pichia pastoris expression vector pPIC9k containing the AOX1 promoter and α-factor signal sequence (between) NotⅠ and SnaBⅠ site .The recombinant plasmid pPIC9kNA was transformed into Pichia pastoris strain GS115. Multi-copy recombinants were screened on concentrations of G418 and Mut^(+) phenotype was confirmed .Multiple insertion transformants were fermented in flasks and induced with (10 mL/L) (methanol). After methanol induction for 4 days, the supernatant was estimated to have antigenicity and high specificity.