摘要
通过反转录PCR从NIH 3T3小鼠成纤维细胞中获得小鼠蛋白激酶CK2 β亚基编码区cDNA ;构建其表达质粒并经测序证明其编码小鼠蛋白激酶CK2 β亚基 ,但与两种已报道的小鼠CK2 β亚基cDNA编码区序列分别存在一个碱基差异 ,与大鼠、猪、兔和人CK2 β亚基cDNA的编码区相应碱基序列则一致。将其在表达菌BL2 1(DE3)中诱导表达 ,出现一 2 6kDa分子量蛋白过度表达 ,表达蛋白占菌体总蛋白的 31 7%,但大多数以不溶形式存在。Westernblot鉴定表明 :过度表达产物能与抗人CK2 β亚基抗体发生特异性免疫反应。当CK2α和 β亚基以 1:1摩尔比混合时 ,构成性的CK2全酶显示出最大活性 ,直接表明CK2 β亚基对CK2α有激活作用。这些结果有力地证明了克隆表达的重组蛋白是小鼠蛋白激酶CK2
The cDNA encoding murine protein kinase CK2β subunit was extracted from NIH 3T3 murine fibroblast cells by RT PCR. The expression plasmid was constructed, then confirmed to encode mouse protein kinase CK2β subunit by DNA sequencing. The sequencing results showed that the sequence of the inserted fragment of two recombinant mouse CK2β clones were differed in the reported two cDNA sequences encoding mouse protein kinase CK2β subunit for one discrepant base respectively, but the difference bases were consistent with the corresponding position of cDNA sequences encoding protein kinase CK2β subunit in rat, rabbit, porcine and human. When the plasmid was induced to express in E.coli BL21(DE3), one protein with molecular mass of 26ku was overexpressed, comprising about 31.7% the total of bacterium protein indicated by scanning. However most of the expressed CK2β proteins were insoluble. Western blot results confirmed that the overexpressed product could specially react with antibody against human CK2β subunit. When recombinant CK2β and α subunits were mixed at an 1:1 molar ratio, the constituted CK2 holoenzyme displayed the maximum activity, which directly illustrated the activation effect of β on α subunit. These results strongly demonstrated that the cloned, expressed recombinant protein was murine protein kinase CK2β subunit.
出处
《基础医学与临床》
CSCD
北大核心
2003年第4期392-398,共7页
Basic and Clinical Medicine
基金
广东省自然科学基金 (0 1176 6 )
广东省科技计划项目 (2 0 0 2C30 10 9)
湛江市科委科技计划项目 (ZK0 0 0 6 )
广东医学院标志性成果扶持项目 (XK0 0 0 2 )
关键词
小鼠
蛋白激酶
CK2β亚基
分子克隆
DNA测序
免疫印迹
regulatory subunit/protein kinase CK2/mouse
expression
molecular cloning
DNA sequence
western blot
