大鼠核转录因子Atoh1的克隆表达及鉴定

Construction of A Recombinant Eukaryotic Vector of Atoh1 and Its Expression in 293-T Cells

郑国玺;祝康;侯瑾;朱珠;韦俊荣;许珉;

1:西安交通大学医学院第二附属医院耳鼻咽喉头颈外病院

摘要
目的利用基因工程方法克隆SD大鼠Atoh1基因CDS区序列,构建大鼠核转录因子Atoh1的真核表达载体并在真核细胞中表达。方法从两只SD大鼠结肠黏膜提取总RNA,采用逆转录PCR法扩增Atoh1基因CDS区序列并亚克隆于PMD-19T载体中。测序鉴定后将Atoh1基因连接于含有EGFP和内部核糖体转入位点(IRES)的真核细胞表达载体pIRES2-EGFP中,对重组质粒进行酶切鉴定和测序鉴定后,以脂质体介导法转染至293T细胞,荧光显微镜和Western blot检测其在293T细胞中的表达。结果扩增得到大鼠Atoh1 CDS区长1 056 bp,编码351个氨基酸,与GeneBank公布的参考序列对比,有两处碱基发生突变,但克隆序列编码的氨基酸序列与参考序列完全一致,两处碱基应为单核苷酸多态性(SNP),突变为无义突变,不影响蛋白表达。双酶切和测序结果证明Atoh1已正确地克隆到真核表达载体pIRES2-EGFP中,荧光显微镜和Western blot证实Atoh1目的蛋白能在293T细胞中稳定表达。结论基因工程方法可成功克隆出Atoh1编码序列,真核表达载体pAtoh1-IRES2-EGFP构建成功并可以在293T细胞中表达。
关键词
感音神经性聋;Atoh1;基因;克隆;真核表达载体
基金项目(Foundation):
国家自然科学基金(NO.30471877);; 陕西省科技攻关项目(NO.2010K15-08)资助
作者
郑国玺;祝康;侯瑾;朱珠;韦俊荣;许珉;
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