Vidalain Pierre-Olivier, Jacob Yves, Hagemeijer Marne C, Jones Louis M, Neveu Grégory, Roussarie Jean-Pierre, Rottier Peter J M, Tangy Frédéric, de Haan Cornelis A M
Unité de Génomique Virale etVaccination, Institut Pasteur, Paris, France,
Methods Mol Biol. 2015;1282:213-29. doi: 10.1007/978-1-4939-2438-7_18.
Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein-protein interaction networks. In the field of virology, it has proven instrumental to identify virus-host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libraries, and only requires a minimum of equipment. We also discuss reasons that motivated our technical choices and compromises that had to be made. This protocol has been used to screen most non-structural proteins of murine hepatitis virus (MHV), a member of betacoronavirus genus, against a mouse brain cDNA library. Typical results were obtained and are presented in this report.
在过去的20年里，酵母双杂交技术成为了解析蛋白质-蛋白质相互作用网络的一项极为重要的技术。在病毒学领域，该技术已被证明有助于识别病毒与宿主之间的相互作用，这些相互作用涉及病毒对细胞功能的利用以及对免疫机制的抑制。在此，我们介绍一种自2006年起就在我们实验室使用的酵母双杂交实验方案，用于寻找300多种病毒蛋白的细胞伴侣。我们的目标是开发一种强大且简便的流程，该流程能将假阳性相互作用降至最低，同时能较好地覆盖目标cDNA文库，并且只需要最少的设备。我们还讨论了促使我们做出技术选择的原因以及不得不做出的权衡。该实验方案已用于针对小鼠脑cDNA文库筛选β冠状病毒属成员小鼠肝炎病毒（MHV）的大多数非结构蛋白。本文报告了获得的典型结果。
