Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells.
Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells.
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基于昆虫细胞中融合依赖性启动子诱导系统测量病毒膜融合蛋白的膜融合活性。
DOI：
10.1099/0022-1317-82-10-2519
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Blissard,GW
中科院分区：
文献类型：
--
作者：
Slack,JM;Blissard,GW
许多病毒膜融合蛋白可以在宿主细胞表面单独表达，然后被触发诱导细胞间融合或合胞体形成。合胞体的形成虽然快速且容易观察，但不容易量化，融合活性的差异也不容易区分或测量。为了解决这个问题，我们开发了一种快速定量的细胞间融合系统，用于比较分析，可能适用于高通量筛选。在这个系统中，报告蛋白，增强型绿色荧光蛋白（EGFP）的表达依赖于细胞间融合。将表达Lac抑制因子- ie1嵌合蛋白的狐尾蛾（Spodoptera frugiperda, Sf9）昆虫细胞在含有响应性合作子启动子的控制下融合到含有egfpreporter构建体的Sf9细胞中。通过所得的EGFP荧光活性测定膜融合效率。以表达假糖橘多衣壳核多角体病毒（OpMNPV） GP64包膜融合蛋白的Sf9细胞为模型进行融合实验。在一个假定的膜融合结构域中，含有单氨基酸取代的GP64蛋白的融合活性发生了细微的变化，EGFP荧光的减少与假定的小膜融合结构域中疏水性的降低相对应。
A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion.Spodoptera frugiperda(Sf9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused toSf9 cells containing anEGFPreporter construct under the control of a responsivelacoperator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity.Sf9 cells expressing theOrgyia pseudotsugatamulticapsid nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.
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影响因子：
3.7
作者：
A. Oomens;S. Monsma;G. Blissard
通讯作者：
A. Oomens;S. Monsma;G. Blissard
影响因子：
5.4
作者：
DOMS, RW;BLUMENTHAL, R;MOSS, B
通讯作者：
MOSS, B
节流阀和阻尼器：控制膜融合引擎
影响因子：
56.9
作者：
Leighninger;D. Robert
通讯作者：
D. Robert
影响因子：
64.8
作者：
Deng, HK;Liu, R;Landau, NR
通讯作者：
Landau, NR
影响因子：
5.4
作者：
MONSMA, SA;BLISSARD, GW
通讯作者：
BLISSARD, GW