CAJ | 학술논문

将绿色荧光蛋白(GFP)连接于蚕豆萎蔫病毒2号(Broad bean wilt virus2,BBWV-2)VP37蛋白的N-端,构建融合基因GFP-VP37,用农杆菌法在BY-2悬浮细胞内进行表达,激光共聚焦显微镜观察融合蛋白的分布.结果显示:GFP-VP37主要定位于细胞核的周围呈网络状,并在细胞边缘形成点状结构;ER-tracker标记显示VP37在细胞内与内质网共定位;电镜免疫金标记显示VP37蛋白主要定位在细胞质中;用BFA处理转染细胞后,VP37在细胞质及细胞边缘的定位受到抑制.推测内质网参与VP37的细胞内转运和分布.
장록색형광단백(GFP)련접우잠두위언병독2호(Broad bean wilt virus2,BBWV-2)VP37단백적N-단,구건융합기인GFP-VP37,용농간균법재BY-2현부세포내진행표체,격광공취초현미경관찰융합단백적분포.결과현시:GFP-VP37주요정위우세포핵적주위정망락상,병재세포변연형성점상결구;ER-tracker표기현시VP37재세포내여내질망공정위;전경면역금표기현시VP37단백주요정위재세포질중;용BFA처리전염세포후,VP37재세포질급세포변연적정위수도억제.추측내질망삼여VP37적세포내전운화분포.
The green fluorescent protein(GFP)was fused to the N-terminal of Broad bean wilt virus 2 (BBWV-2) VP37 protein. The fusion protein was expressed in the BY-2 suspension cells and the green fluorescence was observed using a confocal laser scanning microscope (CLSM). In the cells expressing GFP-VP37 protein, GFP was found located along cell membrane and accumulated around the nuclei. ERTracker was used to label endoplasmic reticulum (ER) . The results showed that the VP37 co-localize with ER. Moreover immuno-gold labeling with VP37 antibody indicated that the VP37 locates in the cytoplasm. After treating with brefildin A (BFA) the fusion protein failed to target plamodesmata. Our observation suggests that the ER was involved in BBWV-2 intracellular transport and distribution.