【摘要】 通过RT PCR从HL 6 0细胞获得人蛋白激酶CK2α′亚基编码区cDNA ,将NdeⅠ HindⅢ双酶切的PCR产物和pT7 7表达载体进行定向克隆、细菌转化、电泳初筛和限制性酶切分析鉴定 .随机挑选阳性克隆进行DNA测序确证 ,筛选含与已知序列完全相符的重组质粒 (命名为pTCKA′) .将其转化BL2 1(DE3)菌 ,IPTG诱导后未见高效特异表达 .然后将人CK2α′cDNA亚克隆至GST融合蛋白表达载体 ,经同样转化和诱导步骤后可见一蛋白特异高效表达 .Western印迹结果证明 :该蛋白能与兔抗人CK2α′3 3 3 3 50 肽段抗血清发生特异性免疫反应 .采用GSH Sepharose 4B柱纯化 ,凝血酶酶切 ,最后从 4g细菌获 4 4mg纯化重组蛋白 .通过性质鉴定和酶动力学分析证明 :克隆、表达和纯化的重组蛋白是有生物学活性的人CK2α′亚基 .更多还原

【Abstract】 The cDNA encoding human protein kinase CK2α′ subunit was obtained from human promyelocytic leukemia cells(HL 60) by RT PCR. Nde Ⅰ/ Hin dⅢ digested PCR product was directionally cloned into pT7 7 expression vector. After transformation, the positive clones were screened out and identified by gel electrophoresis and restriction analysis, respectively. They were selected at random to be sequenced. The clone with sequence identical to that of human CK2α′ cDNA was termed as plasmid pTCKA′. It was transformed into E.coli BL21(DE3) and induced by IPTG, but could not be overexpressed. Then human CK2α′ cDNA was subcloned into an expression plasmid for GST fusion protein. After the same transformation and inducing procedures, one protein was overexpressed. Western blotting confirmed that the overexpressed protein could specifically react with rabbit polyclonal anti human CK2α′ 333 350 antiserum. Purified through Glutathione\|Sepharose 4B affinity chromatography and cutted by thrombin, 4.4mg protein from 4g bacteria was obtained. Characterization and enzyme kinetics of the purified protein demonstrated that the recombinant protein cloned, expressed and purified was human protein kinase CK2α′ with biological activity.更多还原