肝癌中蛋白激酶CK_(2α)亚基的克隆及其功能初步研究

The study of expression and function of CK2α subunit in hepatocellular carcinoma

目的 克隆肝癌中CK2α基因 ,并研究其功能。方法 通过逆转录PCR从肝癌组织中获得CK2α亚基基因编码区cDNA。将NdeI/BamHI双酶切PCR产物连接到同样双酶切并经牛小肠碱性磷酸酶去磷酸化的表达载体 pT7- 7进行定向克隆。转化感受态大肠杆菌DH5获得转化子 ,琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选 4个阳性克隆进行DNA测序 ,将序列完全正确的重组质粒转化表达菌BL2 1(DE3) ,挑单克隆小量培养 ,IPTG诱导表达 ,Westernblot鉴定。结果 转化子的阳性筛选率为 10 0 % ,限制性酶分析结果表明 ,插入片段和重组质粒的大小与理论推测值相符。DNA正反向测序结果表明 :从 4个阳性克隆中筛选到 1个PCR过程不产生突变的重组质粒 ,并将其质粒命名为 pTMCKA。重组质粒转化表达菌经TPTG诱导后出现一个分子量 4 2Kda蛋白过度表达 ,Westernblot结果证明过度表达产物能与抗人CK2α亚基抗体发生特异性免疫反应。结论蛋白激酶CK2α亚基在肝癌中表达 。

Objective To study the expression and function of CK 2α subunit in hepatocellular carcinoma. Methods The aim cDNA was obtained from liver cancer and its paracancerous tissue by RT-PCR.It was directly cloned into pT7-7 expression vector,expressed by inducing withIPTG.The products were identified with Western blotting.Results the expression subunit in liver cancer was higher than that in its paracancerous tissue.The positive rate of transformants was 100%.One protein of 42Kda was overexpressed by inducing with IPTG.The western blot confirmed that the recombinant product could specially react with antibody against human CK 2α subunit.Conclusions CK 2α subunit was expressed in liver cancer and resulted in function.