小鼠SK2基因亚克隆的构建和鉴定

背景:小电导Ca2+激活钾(small conductance Ca2+-activated potassium channels,SK channels)通道存在于大多数可兴奋性细胞并存动作电位的复极化中发挥着重要作用.但SK通道与Ca2+及其他分子的偶合及调控途径尚未明确.目的:为观察SK2通道基因区段与Ca2+及其他分子的偶合及调控,构建pGBAT7和SK2基因片段重组子.方法:根据SK2基因的全长序列,设计3个SK2基因片段引物,经PCR扩增,测序鉴别后,分别亚克隆到酵母表达质粒pGBKT7.构建的pGBKT7-SK2亚克隆通过电转染法分别转染到酵母AH109菌中并被激活.pGBKT7-SK2亚克隆从酵母AH109菌中提取,电泳和测序鉴定.结果与结论:SK2目的基因PCR扩增产物分别为411,546,729 bp.成功构建了3个含有411,546,729 bp的pGBKT7-SK2亚克隆,电泳和测序鉴别表明构建的pGBKT7-SK2亚克隆完全符合设计要求.转染到酵母AH109菌中pGBKT7-SK2亚克隆可被激活,为探讨SK2通道与其他分子的功能相关提供了重要的物质基础.

Construction and identification of mouse SK2 gene subclones

BACKGROUND: Small conductance,Ca2+-activated potassium(SK)channel,presents in various cell types and plays a crucial role in action potential profile.However,coupling and modulation of calcium and associated molecules to SK2 channel remains unclear.OBJECTIVE: To construct the recombinants of pGBAT7 and target fragments of SK2 gene,so as to observe the coupling and regulation of SK2 channel gene to calcium and other molecules.METHODS: Three pairs of primers of the target fragments of SK2 gene were designed and synthesized based on the full-length sequences of SK2.After being identified,they were individually sub-cloned into the yeast expressive plasmid pGBKT7 to construct pGBKT7-SK2 vectors.The recombinant pGBKT7-SK2 vectors were transformed into yeast AH109 by electroporation,and their activation was tested.The recombinants were extracted from yeast AH109 and verified by electrophoresis and sequencing.RESULTS AND CONCLUSION: The target fragments of SK2 gene by PCR were 411,546 and 729 bp,respectively.Three sub-clones of pGBKT7-SK2 were successfully constructed.Electrophoresis and sequencing showed that the constructed sub-clones of pGBKT7-SK2 met the expected requirements.The recombinant pGBKT7-SK2 vectors transformed into the yeast could be activated.The successful construction of the sub-clones of SK2 gene provides an important material basis for further study in the SK2 channel and function-associated molecules.