新加坡石斑鱼虹彩病毒ICP46核输出信号序列的定位与功能鉴定

Definition and function identification of nucleus export signal of SGIV ICP46

新加坡石斑鱼虹彩病毒(SGIV)是一种重要的鱼类传染性病毒,可导致石斑鱼死亡率达90%以上,给石斑鱼的养殖造成巨大的经济损失。SGIV ICP46(infected cell polypeptides 46)是一个立即早期基因,可能参与细胞的生长调控,并对病毒复制有重要作用。在SGIV ICP46序列中存在一段富含亮氨酸(Leucine,L)的潜在核输出信号(nuclear export signal,NES)。为了深入研究该段NES序列在SGIV ICP46核转运过程中的作用,实验构建了3个NES缺失的突变体:仅含NES之前片段的突变体(ΔNESa)、仅含NES之后片段的突变体(ΔNESb)和不含NES的全长片段(ΔNESc),并在真核细胞中表达,观察这些突变体在细胞内的定位情况。转染细胞实验结果表明,野生型EGFP-ICP46重组蛋白可以有效地被输出细胞核,荧光信号主要分布在胞质区;相反,NES缺失的EGFP-ICP46-ΔNES重组蛋白不能被有效地输出细胞核,呈现与EGFP对照相同的泛细胞分布特征。突变实验证明,NES对SGIV ICP46输出细胞核具有决定性作用。

Singapore grouper iridovirus （SGIV） was an important piscine infectious virus. The virus caused more than 90% mortality in grouper and resulted in heavy economic losses in grouper aquaculture. SGIV ICP46 was an immediate-early （IE） gene, may be associated with cell growth control and could contribute to virus replication. A leucine-rich nuclear export signal （NES） in SGIV ICP46 was predicted. To identify the function of the NES in protein translocation, three recombinant plasmids including pEGFP- △NESa （containing the fragment before NES）, pEGFP- ANESb （containing the fargment after NES） and pEGFP- △NESc （containing the complete ICP46 fragment without NES） were constructed and used to track the movement of the protein lacking the NES in GP （Grouper embryo） ceils. As the results showed, in pEGFP-ICP46-transfected cells, the fluorescence signal was observed mainly in the cytoplasm, outside of the nucleus. However, in pEGFP-ICP46-ANES-transfected cells, the fluorescence signal was scattered throughout the whole cell. These results indicated that the NES is essential for the translocation of SGIV ICP46 from the nucleus to the cytoplasm.