大鼠肝tRNA~(Ⅱe)基因在大肠杆菌中的表达及分离纯化

利用T7RNA聚合酶／启动子表达系统在大肠杆菌JM109（DE3）中表达化学合成的大鼠肝tRNAⅡe基因．经酚抽提、DEAE-52离子交换柱层析及HPLC层析，分离纯化大鼠肝tRNAⅡe．变性聚丙烯酰胺凝胶电泳和Northern-blot鉴定表明，大鼠肝tRNAⅡe基因成功地获得表达．氨酸化活性检测表明：纯化后，每1A260单位（40μg）的tRNAⅡe可负载约650pmol的Ⅱe，纯度为54．2％．说明从大肠杆菌中分离纯化出具有一定生物学活性，一定纯度的合成基因表达产物．

Expression and Purification of Rat Liver tRNA~(Ⅱe) Gene Cloned in Eschendis coli

To study the effects of spermine on the construction and function of rat liver tRNAⅡe,a recombinant plasmid containing the synthetic rat liver tRNAⅡe gene was cloned into the lysogen E. coli JM109 (DE3)which contained a single copy of the gene for T7 RNA polymerase in the chromosome under the control of the inducible lacUV5 promoter. By IPTG induction,phenol extracteon and DEAE 52 cellulose chromatography purification,the total E. coli tRNA was isolated and purified. Denaturing PAGE and Northern blot analysis indicated that the rat liver tRNAⅡe gene was successfully expressed in E. coli. The rat liver tRNAⅡe was further purified by the use of Vydac C4 HPLC matrix. Aminoacylation study showed that after HPLC purification, 1A260nm of tRNAⅡe can accept 650 pmol Ⅱe with a purity of 54. 2%.