柔嫩艾美耳球虫真核起始因子3d基因的酵母表达分析
유눈애미이구충진핵기시인자3d기인적효모표체분석
EXPRESSION OF EUKARYOTIC INITIATION FACTOR 3D GENE OF EIMERIA TENELLA IN PICHIA PASTORIS
  • 万方数据
    中国动物传染病学报 중국동물전염병학보
    CHINESE JOURNAL OF VETERINARY PARASITOLOGY
    2012年 2期 47-52 ,共6页

    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

    공춘림%한홍옥%강련련%왕엽%동휘%조기평%주순해%리정%설박%서범범%황병

    球虫%柔嫩艾美耳球虫%真核起始因子3d%毕赤酵母%真核表达 毬蟲%柔嫩艾美耳毬蟲%真覈起始因子3d%畢赤酵母%真覈錶達
    구충%유눈애미이구충%진핵기시인자3d%필적효모%진핵표체
    Coccidia%Eimeria tenella%eIF3d%Pichia pastoris%eukaryotic expression
    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。
    재필적효모진핵표체계통중표체유눈애미이구충진핵기시인자3d（Emeria tenella eukaryotic initiation factor 3d,EteIF3d）기인,병획득구유천연구상적활성단백。장EteIF3d기인극륭도필적효모표체재체pPIC9k상,장구건정학적중조질립통과전격전화입효모세포GS115,용조안산결함배양기화G418분별진행사선,획득함중조질립적효모표체세포。재함1%갑순적BMMY배양기중유도배양,수집1~3 d적표체상청。표체산물진행SDS-PAGE검측,병용Western blot진행면역학분석。결과표명,본연구성공구건료중조질립pPIC9k-EteIF3d,병성공표체해단백,차해단백능여토항EteIF3d혈청특이성결합,구유량호적항원성。EteIF3d기인가재진핵표체계통필적효모중표체,차획득단백구유항원성,종이위진일보연구해단백적결구화공능전정료기출。
    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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