早幼粒细胞白血病基因结构域诱饵载体的构建及鉴定

Construction and Identification of Bait Vector Containing PML Structural Domains

目的研究早幼粒细胞白血病基因（promyelocytic leukemia，PML）中含环指／B—BOX结构与含coiled—coil结构的两个结构域的功能，构建含其结构域序列的诱饵表达载体，为进一步应用酵母双杂交系统筛选与之相互作用的蛋白建立实验基础。方法PCR扩增PML的两个结构域序列，克隆人诱饵载体pG—BKT7中，经测序鉴定后，将诱饵载体转化到酵母细胞AH109中，检测诱饵蛋白有无毒性，渗漏和自激活作用，同时利用蛋白印迹法分析诱饵蛋白的表达。结果成功扩增了PML两个结构域的基因片段，并正确克隆入pGBKT7中。诱饵载体成功转化到酵母细胞AH109中，其中一个诱饵蛋白BD—PML—B无毒性，但具有渗漏和自激活作用，另一个诱饵蛋白BD—PML—C无毒性，渗漏和自激活作用，蛋白印迹法分析证实酵母细胞表达诱饵蛋白。结论含环指／B—BOX的结构域具有转录因子活性，全长PML的转录活性与之有关；成功构建了含coiled—coil结构的PML结合域的酵母诱饵表达载体，为运用酵母双杂交技术筛选与之作用的蛋白并探讨其功能奠定了基础。

Objective To investigate the functions of two structural domains of Promyelocytic leukemia （also known as PML）, ring finger/B-BOX structure and coiled-coil structure, bait expression vector containing the two domains is aimed to construct for screening target proteins interacting with the bait protein by use of yeast two-hybrid system. Methods Fragments of the two structural domain of PML were amplified by PCR, and then were cloned into the bait expression vector pGBKT7. After verified by sequencing, the bait vectors were transformed into AH109 yeast strain. Toxicity, leakage and self-activation of the bait proteins were detected. The expression of the bait protein was analyzed by Western blot. Results The fragments of the two structural donmains of PML were amplified and cloned into pGBKT7 successfully. The resultant bait vectors were transformed into AH109. One of the bait proteins, BD-PML-B, exhibited leakage and self-activation but not toxicity. Another bait protein, BD-PML-C, showed none of toxicity, leakage and self-activation. The expression of either bait protein was confirmed by Western blot. Conclusion The domain of PML with ring finger/B-BOX structure possesses transcription activity and is related to the transcription activity of full-length PML. The domain of PML with coiled-coil structure was constructed successfully, which may be used for screening target proteins interacting with the bait protein by means of yeast two-hybrid technique.