逆转录病毒载体pOZ-KLF5的构建及鉴定

Construction and Characterizations of pOZ-KLF5 Retroviral Plasmid

为研究KLF5的相互作用蛋白从而深入探索其生物功能,构建了C端带有FLAG、HA、6x His三标签的p OZ-KLF5真核表达逆转录病毒载体。p OZ-KLF5载体可通过一次转录得到双顺反子转录单位,即一个转录物能表达两种独立存在的蛋白质:三标签融合蛋白KLF5-3x Tag和筛选标记——白细胞介素-2受体α(IL-2Rα)。通过偶联IL-2Rα抗体的磁珠成功筛选出KLF5-3x Tag表达阳性的293T细胞,经Western-blot检测细胞内KLF5-3x Tag表达情况及其对293T细胞增殖及周期的影响,发现KLF5-3x Tag能够促进细胞克隆的形成并使S期细胞增多,且可被已知E3泛素连接酶WWP1降解,与已有报道一致。进一步通过免疫沉淀实验证明了与KLF5融合的标签的免疫反应性。该质粒的成功构建为研究KLF5相互作用蛋白及深入探索其生物功能奠定了基础。

To analyze KLF5 interacting proteins and explore the biological functions of KLF5, the eukaryotic expression retroviral plasmid pOZ-KLF5, with the FLAG, HA, 6xHis triple tags at C-terminal, was con- structed. Plasmid pOZ-KLF5 can get a bicistronic transcription unit after once transcription and express two independent proteins： triple-tagged fusion protein KLF5-3xTag and selection marker interleukin-2 receptor （IL-2Rα）. 293T cells expressing KLF5-3xTag were successfully selected by the magnetic beads coupling IL-2Rα antibody, and the expression of KLF5-3xTag was detected by Western-blot. Then, the proliferation and cell cycle of 293T were tested, and the results indicated that KLF5-3xTag promotes the proliferation of cell colonies and the G1/S transition. In addition, co-expression of WWPI, a known E3 ligase of KLF5, can decrease KLF5-3xTag protein level, which is consistent with previous reports. Furthermore, immunoprecipi- ration experiment was applied to prove the immunoreaction of the tags. The successful construction of pOZ- KLF5 provides a powerful tool to study the interacting proteins of KLF5 and to explore the biological func- tions of KLF5.