柔嫩艾美耳球虫EtMIC2的酵母表面展示  

Yeast surface display of Eimeria tenella EtMIC2

作　　者：孙慧[1,2] 刘卿[1,2] 张杰[1,2] 陈佩佩[1,2] 王龙江[1,2] 王甜甜[1,2] 王方昆[1,2] 李宏梅[1,2] 肖一红[1] 赵孝民[1,2] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]山东省动物生物工程与疾病防治重点实验室,山东泰安271018

出　　处：《中国兽医学报》2014年第2期231-235,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31172314)

摘　　要：利用酿酒酵母表面展示系统展示柔嫩艾美耳球虫微线蛋白基因EtMIC2,为下一步研制活载体疫苗奠定基础。参照GenBank中柔嫩艾美耳球虫EtMIC2基因序列设计引物,以柔嫩艾美耳球虫的RNA为模板,利用RT-PCR扩增得到预期长度的产物,双酶切连接到酿酒酵母表面展示的载体pCTCON2,转化大肠杆菌TOP10,提取阳性质粒转化酿酒酵母菌株EBY100,诱导表达后,用抗EtMIC2蛋白质特异性抗体,做间接免疫荧光(IFA)检测EtMIC2蛋白的表达。结果显示,EtMIC2成功展示到酵母细胞表面,测得最佳诱导时间为48h。In present study,the Eimeria tenella microneme2 protein （EtMIC2） was designed to be displayed on yeast cell surface using yeast surface display system and explored a screening plat form for developing a living vaccine against chicken coccidiosis. A pair of primers was designed ac cording to the published sequences of EtMIC2 gene encoding the tene[la microneme protein2 deposited in GenBank. We cloned the EtMIC2 gene using first strand eDNA as template after re verse transcripting total RNA from oocysts of E. tenella. Fragment of EtMIC2 gene was ligated into the surface display vector pCTCON2 by double digestion and then transformed with E. coli TOP10. The EtMIC2 expression vector identified by restriction enzyme digestion and sequencing was transformed into EBY100 competent cells. The display of EtMIC2 protein on the yeast surface was detected by indirect immunofluorescence assay using specific antibody against EtMIC2 pro tein. The result showed that EtMIC2 was displayed on the yeast surface and an optimal induction time was 48 h.

关 键 词：柔嫩艾美耳球虫 酵母表面展示 微线蛋白-2 

分 类 号：S852.72[农业科学—基础兽医学]