| 柔嫩艾美耳球虫真核起始因子3d基因的酵母表达分析 |
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| 引用本文： | 孔春林,韩红玉,姜连连,王晔,董辉,赵其平,朱顺海,李婷,薛璞,舒凡帆,黄兵.柔嫩艾美耳球虫真核起始因子3d基因的酵母表达分析[J].中国动物传染病学报,2012(2):47-52. |
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| 作者姓名： | 孔春林 韩红玉 姜连连 王晔 董辉 赵其平 朱顺海 李婷 薛璞 舒凡帆 黄兵 |
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| 作者单位： | 上海师范大学生命与环境科学学院;中国农业科学院上海兽医研究所 农业部动物寄生虫学重点实验室 |
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| 基金项目： | 中央级公益性科研院所基本科研业务费项目(2011JB01) |
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| 摘 要： | 在毕赤酵母真核表达系统中表达柔嫩艾美耳球虫真核起始因子3d(Emeria tenella eukaryotic initiation factor 3d,EteIF3d)基因,并获得具有天然构象的活性蛋白。将EteIF3d基因克隆到毕赤酵母表达载体pPIC9k上,将构建正确的重组质粒通过电激转化入酵母细胞GS115,用组氨酸缺陷培养基和G418分别进行筛选,获得含重组质粒的酵母表达细胞。在含1%甲醇的BMMY培养基中诱导培养,收集1~3 d的表达上清。表达产物进行SDS-PAGE检测,并用Western blot进行免疫学分析。结果表明,本研究成功构建了重组质粒pPIC9k-EteIF3d,并成功表达该蛋白,且该蛋白能与兔抗EteIF3d血清特异性结合,具有良好的抗原性。EteIF3d基因可在真核表达系统毕赤酵母中表达,且获得蛋白具有抗原性,从而为进一步研究该蛋白的结构和功能奠定了基础。
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| 关 键 词： | 球虫 柔嫩艾美耳球虫 真核起始因子3d 毕赤酵母 真核表达 |
EXPRESSION OF EUKARYOTIC INITIATION FACTOR 3D GENE OF EIMERIA TENELLA IN PICHIA PASTORIS |
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| KONG Chun-lin,HAN Hong-yu,JIANG Lian-lian,WANG Ye,DONG Hui,ZHAO Qi-ping,ZHU Shun-hai,LI Ting,XUE Pu,SHU Fan-fan,HUANG Bing.EXPRESSION OF EUKARYOTIC INITIATION FACTOR 3D GENE OF EIMERIA TENELLA IN PICHIA PASTORIS[J].Chinese Journal of Animal Infectious Diseases,2012(2):47-52. |
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| Authors: | KONG Chun-lin HAN Hong-yu JIANG Lian-lian WANG Ye DONG Hui ZHAO Qi-ping ZHU Shun-hai LI Ting XUE Pu SHU Fan-fan HUANG Bing |
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| Institution: | 1.College of Life and Environmental Sciences,Shanghai Normal University,Shanghai 200234,China;2.Key Laboratory of Animal Parasitology,of Ministry of Agriculture,CAAS,Shanghai Veterinary Research Institute,Shanghai 200241,China) |
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| Abstract: | To efficiently express eukaryotic initiation factor 3d(eIF3d) of Eimeria tenella in eukaryotic expression system and obtain the active and natural protein,eIF3d gene was ligated to Pichia pastoris expression vector pPIC9k.The recombinant plasmid was then transformed into yeast cells GS115 by electroporation technology.The positive yeast cells containing recombinant plasmid were screened with histidine-deficient medium and G418.The protein was produced in recombinant yeast cells with induction of 1% methanol in BMMY medium.Supernatant samples were collected from 1 to 3 days.The expressed products were detected in SDS-PAGE and Western blot using rabbit antiserum to eIF3d. |
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| Keywords: | Coccidia Eimeria tenella eIF3d Pichia pastoris eukaryotic expression |
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