酵母双杂试验是一种强大的分子生物学技术，用于研究蛋白质-蛋白质相互作用。它利用转录因子的模块化结构，转录因子有两个独立的域：DNA结合域和激活域。当这两个域被聚合在一起时，它们能够激活基因表达。
酵母双杂试验的原理基于转录因子域的分离。在这个系统中，诱饵蛋白由目标蛋白与DNA结合域融合而成，猎物蛋白由候选蛋白与激活域融合而成。只有当目标蛋白和候选蛋白发生相互作用时，DNA结合域和激活域才会靠近，从而激活报告基因的表达。
酵母双杂试验的实验流程包括五个主要步骤。首先构建诱饵载体和猎物载体，然后将这些载体转化到酵母细胞中。接下来在选择性培养基上筛选阳性克隆，检测报告基因的表达水平，最后验证蛋白质之间的相互作用。这个过程能够有效地识别和确认蛋白质-蛋白质相互作用。
酵母双杂试验在现代分子生物学研究中具有广泛的应用价值。它能够进行高通量的蛋白质相互作用网络筛选，帮助科学家发现新的蛋白质功能和信号通路。在药物开发方面，这项技术也被用于靶点筛选和验证。与其他方法相比，酵母双杂试验操作简便、成本相对较低，特别适用于研究瞬时和弱的蛋白质相互作用。
The principle of yeast two-hybrid assay is based on the separation of transcription factor domains. In this system, the bait protein consists of a target protein fused with a DNA-binding domain, while the prey protein consists of a candidate protein fused with an activation domain. Only when the target protein and candidate protein interact, the DNA-binding domain and activation domain come close together, thereby activating the expression of reporter genes.
The yeast two-hybrid experimental workflow includes five main steps. First, construct bait and prey vectors, then transform these vectors into yeast cells. Next, screen positive clones on selective medium, detect reporter gene expression levels, and finally validate protein-protein interactions. This process can effectively identify and confirm protein-protein interactions.
The mechanism of reporter gene activation in yeast two-hybrid system works as follows: when the target protein and candidate protein interact, the DNA-binding domain binds to the DNA promoter region, while the activation domain is brought close to the promoter. This reconstitutes a functional transcription factor that activates the reporter genes, leading to transcription and production of detectable signals such as growth on selective medium or enzymatic activity.
The yeast two-hybrid assay has broad application value in modern molecular biology research. It enables high-throughput screening of protein interaction networks, helping scientists discover new protein functions and signaling pathways. In drug development, this technique is also used for target screening and validation. Compared to other methods, the yeast two-hybrid assay is simple to operate with relatively low cost, and is particularly suitable for studying transient and weak protein interactions.