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为了解EV71病毒对细胞核转运机制的影响,本研究构建了具有核定位信号的绿色荧光蛋白表达载体(pG-FP-NLS)。将此表达载体转染细胞后,使用EV71病毒感染转染细胞,结果发现EV71病毒可以有效阻止绿色荧光蛋白的核转移。将EV71病毒的2A蛋白酶真核表达载体与pGFP-NLS共转染RD细胞,可以发现2A蛋白酶可阻止具有核定位信号的绿色荧光蛋白的核转移而使绿色荧光蛋白表达于细胞浆。为了进一步研究病毒阻止核转移的机制,病毒感染细胞或通过转染2A蛋白酶真核表达载体进行Nup62的Western blotting检测,结果显示病毒以及2A蛋白酶均可引起Nup62表达下降。证明EV71可通过2A蛋白酶切割Nup62从而抑制核转运。
In order to understand the effect of EV71 virus on nuclear transport mechanism, a green fluorescent protein expression vector (pG-FP-NLS) with nuclear localization signal was constructed in this study. After transfection of this expression vector into cells, the EV71 virus was used to infect transfected cells. As a result, it was found that the EV71 virus can effectively prevent the green fluorescent protein from nuclear transfer. The co-transfection of the EV71 virus 2A protease eukaryotic expression vector with pGFP-NLS to RD cells revealed that 2A protease blocked nuclear translocation of the green fluorescent protein with nuclear localization signals and led to green fluorescent protein expression in the cytoplasm. To further investigate the mechanism by which the virus blocks nuclear translocation, viral infection of cells or detection of Nup62 by transfection with the 2A protease eukaryotic expression vector revealed that both the virus and the 2A protease caused a decrease in Nup62 expression. EV71 demonstrated that EV71 can inhibit nuclear translocation by cleaving Nup62 by 2A protease.