腸道病毒71型3D聚合酶轉(zhuǎn)錄激活域的界定
發(fā)布時間：2018-11-28 14:50
【摘要】：EV71是導(dǎo)致手足口病的主要病原體之一,其3D聚合酶作為依賴于RNA的RNA聚合酶,在病毒基因組轉(zhuǎn)錄及復(fù)制過程中發(fā)揮重要作用。當前,3D與宿主細胞的相互作用鮮有研究。酵母雙雜交實驗是研究蛋白質(zhì)相互作用成熟有效的方法。本文將3D基因克隆至pGBKT7載體,構(gòu)建了用于酵母雙雜交實驗的誘餌質(zhì)粒,鑒定正確后轉(zhuǎn)化酵母細胞AH109,依次檢測了3D蛋白的表達、細胞毒性和自激活能力,結(jié)果表明3D基因可在AH109菌株中表達,對后者生長無顯著影響,但融合蛋白具有自激活能力。進一步構(gòu)建一系列含3D蛋白截短體的誘餌質(zhì)粒,通過自激活實驗界定了3D的最小轉(zhuǎn)錄激活域(1~94aa),為后續(xù)利用酵母雙雜交技術(shù)研究與3D相互作用的細胞蛋白奠定了基礎(chǔ)。
[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.
【作者單位】： 南開大學(xué)分子微生物與技術(shù)教育部重點實驗室南開大學(xué)生命科學(xué)學(xué)院;
【基金】：“973”計劃(項目號:2013CB911100),題目:重要病毒轉(zhuǎn)錄復(fù)制蛋白復(fù)合體的結(jié)構(gòu)功能研究
【分類號】：R373.2
本文編號：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.
【作者單位】： 南開大學(xué)分子微生物與技術(shù)教育部重點實驗室南開大學(xué)生命科學(xué)學(xué)院;
【基金】：“973”計劃(項目號:2013CB911100),題目:重要病毒轉(zhuǎn)錄復(fù)制蛋白復(fù)合體的結(jié)構(gòu)功能研究
【分類號】：R373.2
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