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目的利用酵母双杂交技术筛选与人巨细胞病毒US28蛋白相互作用的宿主蛋白,可为研究US28蛋白的作用机制提供依据。方法构建p GBKT7-US28诱饵重组载体,检测其在酵母细胞中的表达和自激活作用,然后利用酵母双杂交系统筛选人脑文库中与US28相互作用的蛋白质。对获得的阳性克隆进行PCR鉴定、测序及序列比对分析,并通过酵母共转化实验再次确认蛋白的相互作用。结果 p GBKT7-US28在酵母中成功表达US28融合蛋白;酵母双杂交筛选并验证,获得了5个与US28蛋白相互作用的宿主蛋白。结论初步鉴定得到5个与US28相互作用的细胞蛋白,为探索US28蛋白的新功能以及揭示巨细胞病毒的致病机理和疾病治疗奠定基础。
Objective To screen the host proteins interacting with human cytomegalovirus US28 by yeast two-hybrid technique, and to provide basis for studying the mechanism of action of US28 protein. Methods The pGBKT7-US28 bait recombinant vector was constructed and its expression and self-activation in yeast cells were detected. Then the yeast two-hybrid system was used to screen the proteins interacting with US28 in the human brain library. Positive clones obtained were identified by PCR, sequencing and sequence alignment analysis, and protein interaction was confirmed again by yeast cotransformation assay. Results pGBKT7-US28 successfully expressed US28 fusion protein in yeast. After yeast two-hybrid screening and validation, five host proteins interacting with US28 protein were obtained. Conclusion Preliminary identification of five cell proteins that interact with US28 laid the foundation for exploring new functions of US28 protein and revealing the pathogenesis and disease treatment of cytomegalovirus.