重组猪蛔虫抗菌肽cecropin P1在毕赤酵母中的表达及抑菌活性  

Expression of Ascaris suum cecropin P1 gene in Pichia pastoris and its antimicrobial activity

作　　者：李巍[1] 仲飞[1] 李秀锦[2] 张峰[1] 刘龙飞[1] 王淼[3] 李凌[1] 王幸兴[1] 韩冬梅[1] 潘素敏[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071001 [2]燕山大学环境与化学工程学院生物工程系,河北秦皇岛066004 [3]保定市畜牧水产局,河北保定071051

出　　处：《中国兽医学报》2011年第8期1133-1137,共5页Chinese Journal of Veterinary Science

基　　金：河北省科技支撑计划资助项目(0722401D);河北农业大学校长基金资助项目(XZJJ2005-06)

摘　　要：根据已发表的蛔虫抗菌肽cecropin P1基因编码序列设计引物,采用RT-PCR方法从猪肠道蛔虫中扩增出cecropin P1基因,并将其插入到酵母分泌型表达载体pPIC9K中α-因子信号肽下游的SnaBⅠ和NotⅠ位点之间,构建成cecropin P1基因的酵母分泌型表达载体pPIC9K/cecropin P1。载体经SalⅠ酶切线性化,电转化到组氨酸缺陷型的酵母宿主菌GS115中,然后利用以葡萄糖为碳源的培养基(MD)和以甲醇为碳源的培养基(MM)筛选出组氨酸His+型和甲醇利用正型(Mut+)酵母重组体,再经G418加压筛选得到高拷贝cecropin P1基因的重组酵母。经PCR检测证明cecropin P1基因已整合到毕赤酵母的染色体中。重组酵母经摇瓶发酵培养和甲醇诱导,通过Tricine-SDS-PAGE检测,重组酵母能够分泌表达重组cecropin P1,同时表达的cecropin P1对大肠杆菌和金黄色葡萄球菌均有明显的抑菌活性。To express Ascaris suum antimicrobial peptide cecropin P1 in Pichia pastoris,the primers was designed according to published cDNA sequence of cecropin P1 and amplified cecropin P1 cDNA from Ascaris suum by RT-PCR.The cecropin P1 gene was cloned into yeast shuttle vector pPIC9K down stream of α-factor signal peptide sequence by SnaBⅠand NotⅠsites to generate the recombinant secretion vector pPIC9K/cecropin P1.The recombinant vector,linearized by SalⅠ,was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation.We selected His＋-transformed methylotropic（His＋,Mut＋） yeast using histidine-absent medium containing dextrose（MD） or methanol（MM） as the only carbon source,and then screened the recombinant GS115 with multi-copy cecropin P1 genes by G418.The recombinant yeast encoding cecropin P1 was identified by PCR.The results showed that cecropin P1 gene was integrated into yeast genome.The secretive expression of cecropin P1 was performed under the induction of methanol in shaking flask culture.The recombinant yeast can generate secreting expression of cecropin P1 identified by Tricine-SDS-PAGE.The recombinant cecropin P1 has the antibacterial activity against Escherichia coil and Staphylococcus aureus.

关 键 词：猪蛔虫 抗菌肽 CECROPIN P1 毕赤酵母 抑菌活性 

分 类 号：S852.7[农业科学—基础兽医学]