酵母双杂交系统检测SK2与RyR2蛋白的相互作用

Identification of interaction between SK2 and RyR2 protein by yeast two-hybrid system

目的:探讨小电导Ca2+激活K+(SK)通道亚型SK2通道与肌浆网(RyR)2蛋白的相互作用。方法:经PCR扩增及酶切鉴定,构建含有SK2和RyR2目的基因片段的酵母表达载体pGBKT7-SK2和pGADT7-RyR2。pG-BKT7-SK2与pGADT7-RyR2经电转化法转化酵母宿主AH109,X-α-Gal/3AT/SD/-Trp-Leu培养鉴定阳性克隆。结果:获得3个重组质粒pGBKT7-SK2和16个重组子pGADT7-RyR2。酵母双杂交检测结果显示pGBKT7-SK2与pGADT7-RyR2可转化酵母AH109,但无阳性菌落生长。结论:SK2和RyR2蛋白之间可能无直接相互作用。

Aim：To detect the interaction of small-conductance calcium-activated potassium channel（SK）2 with ryanodine receptor（RyR）2.Methods：The yeast expressive plasmid pGBKT7 vectors containing target fragments of SK2 gene and the pGADT7 vectors with the fragments of RyR2 gene were individually constructed by PCR and restricted endonuclease.The recombinants of pGBKT7-SK2 and pGADT7-RyR2 were co-transformed into yeast AH109 by electrophoration and inoculated into the X-α-Gal/3AT/SD/-Trp-Leu plate.The positive colonies in the plates were screened by X-α-Gal color.Results：Three recombinants of pGBKT7-SK2 and sixteen recombinants of the pGADT7-RyR2 vectors were successfully constructed and transformed into the yeast host AH109.But no positive clonies in the plates were identified.Conclusion：The interaction of SK2 with RyR2 protein has not been detected in the yeast.