重组人蛋白激酶CK2β亚基cDNA的克隆与测序

Cloning and Sequencing of Recombinant cDNA Encoding Human Protein Kinase CK2 β Subunit

蛋白激酶ＣＫ２是一种存在的信使非依赖性丝／苏氨酸蛋白激酶．它是由两个催化亚基（α或α′）和两个调节亚基（β）组成的不均一四聚体．用反转录ＰＣＲ从ＨＬ－６０细胞中获得了人蛋白激酶ＣＫ２β亚基编码区ｃＤＮＡ，将ＮｄｅⅠ／ＨｉｎｄⅢ双酶切ＰＣＲ产物连接到表达载体ｐＴ７－７的ＮｄｅⅠ／ＨｉｎｄⅢ双酶切位点中．转化感受态细菌ＤＨ５α获得转化子，阳性筛选率为７２％．限制性酶切分析结果表明，插入片段和重组质粒的大小与理论推测值相符．随机挑选４个阳性质粒测定其插入片段ＤＮＡ序列，结果显示有２个含有正确插入的人蛋白激酶ＣＫ２βｃＤＮＡ，命名为ｐＴＣＫＢ．其余２个克隆分别存在１个和２个点突变，即在其编码区ｃｏｎｄｏｎ１４８的ＴＣＡ→ＴＴＡ，结果Ｓｅｒ→Ｌｅｕ；另一个则在Ｃｏｎｄｏｎ１４３ＧＴＧ→ＡＴＧ，Ｖａｌ→Ｍｅｔ；Ｃｏｎｄｏｎ１７０ＧＴＧ→ＧＣＧ，Ｖａｌ→Ａｌａ．重组质粒（ｐＴＣＫＢ）克隆的成功，将为在原核细胞中表达蛋白激酶ＣＫ２β亚基以及利用ＣＫ２βｃＤＮＡ作为探针进行深入研究打下基础．

Protein kinase CK2,formerly known as casein kinase 2 or CKII,is a ubiquitous signal independent Ser/Thr protein kinase.It is a heterotramer composed of two catalytic subunits (α or α′) and two regulatory (β) subunits (α 2β 2,αα′β 2,α′ 2β 2).The cDNA encoding human protein kinase CK2 β subunit was obtained from human promyelocytic leukemia cells (HL 60) by RT PCR. NdeⅠ/Hin dⅢ digested PCR products was ligated into the NdeⅠ/Hin dⅢ digested restriction site of expressor vector pT7 7.After transformation of the competent E.coli DH5α,transformants were obtained and isolated.The positive rate of screening was 72%.The result of restriction analysis indicated that DNA band size of the insert fragement and recombinant plasmid were consistent with theoretical predicated values.The DNA of insert fragements of four positive clones selected at random were sequenced.The result showed that two clones possessed correct sequence of encoding region of human protein kinase CK2 β subunit, termed as pTCKB;the other two contained one (TCA→TTA,Ser 140 →Leu) and two (GTG→ATG,Val 143 →Met;GTG→GCG,Val 170 →Ala) point mutant,respectively.This recombinant plassmid (pTCKB) expressed human protein kinase CK2 β subunit in prokaryocyte.CK2β cDNA in this plasmid was used as a probe and bait. Successful cloning of pTCKB laid a solid basis for further study and built a set of successful methods for constructing other recombinant plamids by using the expressor vector pT7 7.