Evaluation of VP22 spread in tissue culture
Evaluation of VP22 spread in tissue culture
复制标题
DOI：
10.1128/jvi.74.2.1051-1056.2000
复制
发表时间：
2000-01-01
影响因子：
5.4
通讯作者：
O'Hare, P
中科院分区：
文献类型：
--
作者：
Brewis, N;Phelan, A;O'Hare, P
我们比较单纯疱疹病毒蛋白VP 22和绿色荧光蛋白（GFP）-VP 22融合蛋白的细胞间转运的检测方法。使用有机固定剂通过免疫荧光（IF）观察两种蛋白质的扩散。这两种蛋白质的传播也检测到多聚甲醛（PFA）固定和洗涤剂透化后，IF，虽然在降低的水平。然而，虽然通过检查甲醇固定后的固有GFP荧光观察到GFP-VP 22的扩散，但PFA固定后几乎没有观察到扩散，这表明受体细胞中融合蛋白的水平低于固有荧光的检测限，或者PFA固定淬灭了GFP-VP 22的荧光。我们进一步考虑了VP 22从甲醇固定的细胞中洗脱和固定后与周围细胞的结合是否有助于甲醇固定后观察到的扩散检测增加。结果表明，虽然这可能发生，但似乎是一种轻微的影响，无法解释培养物中观察到的VP 22细胞间扩散。
We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.