AcMNPV e18基因酵母双杂交诱饵载体构建和转录自激活检测

Construction of an Yeast Two-Hybrid Bait Vector of Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) and Testing of Autonomous Transcriptional Activation

用PCR方法扩增苜蓿银纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhe-drovirus,AcMNPV)被膜蛋白ODV-E18基因,克隆至酵母双杂交诱饵载体pGBKT7构建诱饵质粒pG-BKT7-e18。将诱饵质粒分别转化酵母菌株Y187和AH109感受态细胞,被转化细胞在涂有X-gal的SD/-Trp营养缺陷型固体培养基上形成白色菌落;在SD/-Trp/-His和SD/-Trp/-Ade固体培养基上均不形成菌落,表明诱饵基因表达产物BD-E18在这两种细胞中都不能激活报告基因转录。pGBKT7-e18转化的Y187细胞在SD/-Trp营养缺陷型液体培养基中的生长速度与空载体转化细胞相同,显示BD-E18对酵母细胞无细胞毒性。结果表明,AcMNPV ODV-E18可能不直接参与对宿主细胞或病毒基因表达的调节,其编码基因可以作为诱饵基因通过筛查病毒宿主cDNA文库识别与其相互作用的蛋白质。

The DNA sequence encoding an envelope protein,ODV-E18,of Autographa californica multiple nucleopolyhedrovirus(AcMNPV) was amplified by PCR and cloned into pGBKT7 to construct bait plasmid pGBKT7-e18 for yeast two-hybrid screening.The bait plasmid was used to transform yeast strains Y187 and AH109 respectively for assays on cytotoxity and autonomous transcriptional activation.Both transformed Y187 and AH109 cells formed white colonies on the plates with auxotroph SD /-Trp medium and X-gal,but could not grow on the plates with auxotroph SD /-Trp /-His or SD /-Trp /-Ade medium,showing that the BD-E18 encoded by the bait plasmid could not activate transcription of the reporter genes.The Y187 cells transformed by the bait plasmid grew as fast as the ones transformed with the empty vector,indicating that BDE18 was nontoxic to the cells.The results suggested that AcMNPV ODV-E18 is unlikely involved in regulation on transcription of host or virus genes,and that the coding sequence of E18 could be used as bait to screen a cDNA library of host insect for identification of proteins interacting with the viral protein.