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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在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411,546,729bp。成功构建了3个含有411,546,729bp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。
BACKGROUND: Small conductance Ca2 + -activated potassium channels (SK channels) exist in most excitable cells and play an important role in the repolarization of action potentials. However, the pathway of coupling and regulation of SK channel with Ca2 + and other molecules is not yet clear. OBJECTIVE: To observe the coupling and regulation of SK2 channel gene segment with Ca2 + and other molecules, and to construct recombinant pGBAT7 and SK2 gene fragments. Methods: Three SK2 gene fragments were designed according to the full-length sequence of SK2 gene. After PCR amplification and sequencing, they were subcloned into the yeast expression plasmid pGBKT7. The constructed pGBKT7-SK2 subclones were respectively transfected into yeast AH109 by electroporation and activated. The pGBKT7-SK2 subclone was extracted from the yeast strain AH109 and identified by electrophoresis and sequencing. RESULTS AND CONCLUSION: The PCR products of SK2 gene were 411,546,729 bp respectively. Three pGBKT7-SK2 subclones containing 411,546,729bp were successfully constructed. The electrophoresis and sequencing showed that the constructed pGBKT7-SK2 subclones fully met the design requirements. The pGBKT7-SK2 subclone transfected into yeast AH109 can be activated, providing important material basis for exploring the function of SK2 channel and other molecules.