【摘要】 通过PCR扩增构建的重组质粒pTHCKA′获得人蛋白激酶CK2α′亚基编码区cDNA ,将PstⅠ NdeⅠ双酶切的PCR产物 ,连接到同样酶切的“诱饵”载体pGBKT7,构建为酵母双杂交穿梭表达质粒 (BD质粒 ) ,经DNA测序确证及转染感受态酵母菌AH10 9以排除自主激活 .从体外培养的HL 6 0细胞中提取总RNA ,采用CLONTECHSMART(switchingmechanismat 5′endofRNAtranscript)技术 ,在酵母细胞中构建HL 6 0细胞的酵母双杂交cDNA文库 ,将构建的BD质粒、文库cDNA、酵母穿梭表达质粒pGADT7 Rec ,共转染感受态酵母菌AH10 9进行酵母双杂交实验 .筛选与人蛋白激酶CK2α′相互作用蛋白 .筛选结果及一对一回复性实验显示 ,有 6种与CK2α′相互作用蛋白 ,核酸序列分析及同源性检索表明 ,其中 1种与醛缩酶A有高度同源性 (99 6 % ) .更多还原

【Abstract】 The target CK2α′ cDNA was obtained by amplifying the recombinant plasmid pTHCK? ?containing human protein kinase CK2α′ subunit cDNA with PCR. PstⅠ/NdeⅠ-digested PCR products were directionally cloned into DNA-BD vector pGBKT7 which had been digested by PstⅠ/NdeⅠ. The recombinant plasmid is a yeast two-hybrid BD bait plasmid which followed by DNA sequencing and auto-activated experiment. Total RNA was extracted from HL-60 cells using CLONTECH SMART method, and a cDNA library was successfully constructed in the yeast cell. The BD plasmid, library cDNA and pGADT7-Rec plasmid were cotransformed into the competent AH109. After yeast two-hybrid experiment and positive clones were retested by cotransformation, six interaction proteins with human protein kinase CK2α′ subunit were screened. The sequence analysis and homology comparison showed that one of the interaction proteins was highly homologous with aldolase A(99.6%).更多还原