摘要
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.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第2期228-231,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
广东省自然科学基金
广东省卫生厅青年科研基金
关键词
人蛋白激酶CK2
Β亚基
DNA测序
重组
CDNA
Human protein kinase CK2,β Subunit,Expressor vector pT7 7,Cloning,DNA sequencing