PRSV运动相关病毒蛋白的Sos恢复系统诱饵载体构建及自激活效应检测

为研究与番木瓜环斑病毒运动相关的病毒蛋白(CP、HC-Pro、CI)互作的寄主因子,构建以Sos恢复系统为原理的胞质酵母双杂交系统的3个病毒蛋白诱饵载体,并检测其自激活作用来验证是否适用于后续的文库筛选.通过RT-PCR从番木瓜环斑病毒海南分离物中扩增出这3个病毒蛋白的片段,按正确方向将目标片段分别插入诱饵载体pSos中,并将重组质粒导入酵母温度敏感酵母菌株cdc25H,检测其表达产物对酵母Sos恢复系统Ras信号通路的激活作用而产生的细胞温敏特性影响.结果表明,这3个含番木瓜环斑病毒运动相关的病毒蛋白的诱饵载体构建正确,对酵母菌株cdc25H的Sos恢复系统无激活作用,激活试验为阴性.

Construction and Verification of Bait Vectors Containing Viral Proteins Relating to Papaya Ringspot Virus Movement in Sos Recruitment System

A study was made to observe the interactions between viral proteins relating to papaya ringspot virus movement and Carica papaya proteins. The CP, HC-Pro and CI relating to PRSV movement were cloned by RT-PCR from Papaya ringspot virus(Hainan isolate). These viral genes were fused with the Sos gene of the plasmid pSos to construct bait vectors of a Sos recruitment system respectively. PCR and sequencing analysis showed these bait vectors were successfully constructed. The bait vectors were then transformed into the temperature-sensitive strain cdc25H and tested for their effect on the activation of the Sos recruitment system respectively. The activation and suitability testing showed negative results. It is concluded these bait vectors can be used to screen Carica papaya proteins interacting with viral proteins relating to papaya ringspot virus movement from cDNA library.