| 柔嫩艾美耳球虫MIC4-N的真核表达 |
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| 引用本文： | 王卫婷,姚鹏,周赛,王黎霞,安健.柔嫩艾美耳球虫MIC4-N的真核表达[J].北京农学院学报,2011,26(3):24-27. |
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| 作者姓名： | 王卫婷 姚鹏 周赛 王黎霞 安健 |
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| 作者单位： | 1. 北京农学院动物科学技术学院,北京,102206
2. 北京农业职业学院,北京,102442 |
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| 基金项目： | 国家自然科学基金资助项目 |
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| 摘 要： | 应用RT-PCR技术扩增柔嫩艾美耳球虫的MIC4-N端基因并去掉信号肽,为了表达检测和纯化的方便,设计引物时改变了3＇端的终止密码子,使MIC4-N的C末端带有c-myc和6His抗原标签。通过双酶切,使加工好的MIC4-N基因连接到毕赤酵母表达载体pPICZαA上,转化JM109感受态细胞。再经EcoRⅠ与NotⅠ双酶切、菌落PCR及测序鉴定,证明目的基因与真核表达载体pPICZαA构建成功,单酶切重组表达载体、电转转入GS115酵母感受态细胞中。用含高浓度Zeocin的YPD平板筛选酵母菌落,菌落PCR法鉴定转化成功的阳性菌,在BMMY培养基中摇瓶培养,并用甲醇诱导,经SDS-PAGE及Western Blot鉴定,蛋白分泌表达成功。
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| 关 键 词： | 柔嫩艾美耳球虫 微线体蛋白4-N 毕赤酵母真核表达 |
Eukaryotic expression of Eimeria tenella MIC4-N |
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| WANG Wei-ting,YAO Peng,ZHOU Sai,WANG Li-xia,AN Jian.Eukaryotic expression of Eimeria tenella MIC4-N[J].Journal of Beijing Agricultural College,2011,26(3):24-27. |
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| Authors: | WANG Wei-ting YAO Peng ZHOU Sai WANG Li-xia AN Jian |
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| Institution: | (1.College of Animal Science and Veterinary Medicine,Beijing University of Agriculture,Beijing 102206,China;2.Beijing Vocational College of Agriculture,Beijing 102442,China) |
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| Abstract: | The sequences of EtMIC4-N gene were amplified by RT-PCR and deleted N terminal signal peptide.Meanwhile,in order to facilitate detection and purification of the expression,the designation of primers for changing the 3 ’end of the stop codon was carried out,and the target protein was labeled with a C-terminal c-myc and 6His antigen.The processed MIC4-N gene was connected to pPICZαA,an expression vector of Pichia Pastoris by double digestion and transformed into JM109 competent cells.The recombinant expression vectors being identified by double digestion,PCR and sequencing were linearized and transformed into GS115 competent cells.After being screened by YPD containing high concentrations of Zeocin and PCR,the positive clone was cultured in flask with BMMY and induced by methanol.The results of SDS-PAGE and Western Blot analysis suggested that this recombinant protein was successfully expressed and secreted into BMMY supermatant. |
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| Keywords: | Eimeria tenella EtMIC4-N Eukaryotic expression of pPICZαA |
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