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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
