| 大鼠核转录因子Atoh1的克隆表达及鉴定 |
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| 引用本文： | 郑国玺,祝康,侯瑾,朱珠,韦俊荣,许珉. 大鼠核转录因子Atoh1的克隆表达及鉴定[J]. 听力学及言语疾病杂志, 2011, 19(5): 438-443. DOI: 10.3969/j.issn.1006-7299.2011.05.015 |
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| 作者姓名： | 郑国玺 祝康 侯瑾 朱珠 韦俊荣 许珉 |
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| 作者单位： | 西安变通大学医学院第二附属医院耳鼻咽喉头颈外病院(西安710004) |
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| 基金项目： | 国家自然科学基金，陕西省科技攻关项目 |
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| 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。
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| 关 键 词： | 感音神经性聋 Atoh1 基因 克隆 真核表达载体 |
Construction of A Recombinant Eukaryotic Vector of Atoh1 and ItsExpression in 293-T Cells |
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| Zheng Guoxi,Zhu Kang,Hou Jin,Zhu Zhu,WeiJunrong,Xu Min. Construction of A Recombinant Eukaryotic Vector of Atoh1 and ItsExpression in 293-T Cells[J]. Journal of Audiology and Speech Pathology, 2011, 19(5): 438-443. DOI: 10.3969/j.issn.1006-7299.2011.05.015 |
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| Authors: | Zheng Guoxi Zhu Kang Hou Jin Zhu Zhu WeiJunrong Xu Min |
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| Affiliation: | Zheng Guoxi,Zhu Kang,Hou Jin,Zhu Zhu,Wei Junrong,Xu Min(Department of Otolaryngology-Head and Neck Surgery,the Second Affiliated Hospital,Medical School of Xi'an Jiaotong University,Xi'an,71004,China) |
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| Abstract: | Objective Useing gene engineering technique to clone SD rats Atoh1 cDNA coding sequence and construct the Eukaryotic expression vector for its expression in eukaryotic cells.Methods Total RNA was extracted from colon of SD rats,by means of asymmetrical primer/template,double stranded cDNA of Atoh1 was gained,then the cDNA coding sequence was cloned into PMD-19T vector and sequenced.The purified digested fragment was connected into Eukaryotic expression vector pIRES2-EGFP with the EGFP gene and the internal ... |
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| Keywords: | Sensorineural hearing loss Atoh1 Gene Clone Eukaryotic expression vector |
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