肠道病毒71型3D聚合酶转录激活域的界定
发布时间:2018-11-28 14:50
【摘要】:EV71是导致手足口病的主要病原体之一,其3D聚合酶作为依赖于RNA的RNA聚合酶,在病毒基因组转录及复制过程中发挥重要作用。当前,3D与宿主细胞的相互作用鲜有研究。酵母双杂交实验是研究蛋白质相互作用成熟有效的方法。本文将3D基因克隆至pGBKT7载体,构建了用于酵母双杂交实验的诱饵质粒,鉴定正确后转化酵母细胞AH109,依次检测了3D蛋白的表达、细胞毒性和自激活能力,结果表明3D基因可在AH109菌株中表达,对后者生长无显著影响,但融合蛋白具有自激活能力。进一步构建一系列含3D蛋白截短体的诱饵质粒,通过自激活实验界定了3D的最小转录激活域(1~94aa),为后续利用酵母双杂交技术研究与3D相互作用的细胞蛋白奠定了基础。
[Abstract]:EV71 is one of the major pathogens leading to HFMD. As a RNA dependent RNA polymerase, 3D polymerase plays an important role in viral genome transcription and replication. At present, there is little research on the interaction between 3D and host cells. Yeast two-hybrid experiment is an effective method to study protein interaction maturation. In this paper, the 3D gene was cloned into pGBKT7 vector, and the bait plasmid was constructed for yeast two-hybrid experiment. After identified correctly, the expression of 3D protein, cytotoxicity and self-activation ability of transformed yeast cell AH109, were detected in turn. The results showed that 3D gene could be expressed in AH109 strain and had no significant effect on the growth of the latter, but the fusion protein had the ability of self-activation. A series of bait plasmids containing 3D protein truncated bodies were further constructed, and the 3D minimum transcriptional activation domain (1~94aa) was defined by self-activation experiments, which laid a foundation for the further study of cellular proteins interacting with 3D by yeast two-hybrid technique.
【作者单位】: 南开大学分子微生物与技术教育部重点实验室南开大学生命科学学院;
【基金】:“973”计划(项目号:2013CB911100),题目:重要病毒转录复制蛋白复合体的结构功能研究
【分类号】:R373.2
本文编号:2363187
[Abstract]:EV71 is one of the major pathogens leading to HFMD. As a RNA dependent RNA polymerase, 3D polymerase plays an important role in viral genome transcription and replication. At present, there is little research on the interaction between 3D and host cells. Yeast two-hybrid experiment is an effective method to study protein interaction maturation. In this paper, the 3D gene was cloned into pGBKT7 vector, and the bait plasmid was constructed for yeast two-hybrid experiment. After identified correctly, the expression of 3D protein, cytotoxicity and self-activation ability of transformed yeast cell AH109, were detected in turn. The results showed that 3D gene could be expressed in AH109 strain and had no significant effect on the growth of the latter, but the fusion protein had the ability of self-activation. A series of bait plasmids containing 3D protein truncated bodies were further constructed, and the 3D minimum transcriptional activation domain (1~94aa) was defined by self-activation experiments, which laid a foundation for the further study of cellular proteins interacting with 3D by yeast two-hybrid technique.
【作者单位】: 南开大学分子微生物与技术教育部重点实验室南开大学生命科学学院;
【基金】:“973”计划(项目号:2013CB911100),题目:重要病毒转录复制蛋白复合体的结构功能研究
【分类号】:R373.2
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