单质粒哺乳动物单杂交系统的构建及功能评价
Development and functional evaluation of single-plasmid mammalian one-hybrid system

对双质粒单杂交系统进行改进研究，将两种质粒的功能进行融合，最终得到单质粒杂交系统，并利用HEK 293T细胞对融合质粒进行了功能测试和评价. 研究结果表明：以RXRα为基础进行活性测试时，该系统对激动剂/拮抗剂响应的灵敏度与双质粒系统相近；该系统也适用于ERα、GRα、RARα、LXRα、PPARγ等多种核受体的活性检测，当添加已知配体时，其激活倍数介于8~36倍之间，Z因子大于0.6，可以作为配体筛选的基本模型；以Nur77为基础进行实验精密度测试时，单质粒系统的CV值较双质粒系统有显著降低，体现出更为优异的精确度和稳定性. 因此，该模型在核受体潜在配体的筛选过程中能够提供更为简便的操作和准确的结果，具有良好的应用前景.
The traditional dual-plasmid one-hybrid system was improved by fusing the essential sequences together，thus to obtain a single-plasmid one-hybrid system. When tested in HEK 293T cells，the results showed that after transfection of specified plasmid，the response of RXRα observed in single-plasmid system to the agonist/antagonist was similar to that of dual-plasmid system. The system could also be applied to the ligand screening of ERα，GRα，RARα，LXRα and PPARγ. When known ligands were added，the activation ratios of these NRs were between 8 and 36 folds with Z factors more than 0.6，thus it could serve as a basic screening model. Furthermore，the CV of single plasmid system was significantly lower than that of dual-plasmid system when Nur77 was used for precision evaluation. Indicating that the single-plasmid system possesses both sensitivity and stability. Therefore，the new system is convenient，robust and sensitive，which makes it promising in ligand screening of NRs