【摘要】 <正> T4-RNA连接酶自1972年由Silber等人发现以来,已有不少的研究结果说明,T4-RNA连接酶在没有模板的情况下,能有效地催化RNA5′-端磷酸与3′-端羟基形成分子内或分子间的磷酸二酯键。它亦催化DNA之间,或RNA与DNA之间的连接反应。在酶的分离纯化方面,亦相继进行了不少的研究工作,并有了改进,可以比较容易地获得较大量的一定纯度的制品,给研究工作提供方便与可能。它是一种有用的工具酶被用来合成具有确定顺序的多核苷酸更多还原

【Abstract】 T4-RNA ligase has been extensively used in the synthesis of nucleic acid with defined seqences. We prepared an immobilized T4-RNA ligase by coupling the enzyme to activated CH-Sepharose 4B.Its capacity is about 0.3 unit of RNA ligase per milligram immobilized enzyme and it can be used in joining reaction. The immobilized) enzyme catalyzed the exchange reaction between ATP and 32ppt.It catalyzed the addition of nucleoside 3’ ,5’-diphosphates to oligonucleotide.lt also catalyzed the-intermolecular joining of oligonucleotides,or oligonucleotide and tRNA.In some of the reactions with smaller oligonucleotides as substrates,the yields of joining products were as good as those that the reactions were catalyzed by soluble T4-RNA. ligase.The immobilized enzyme was packed into a small column and was used repeatedly to catalyze the joining reaction. At sixth time,the activity of the immobilized enzyme did not decrease.These preliminary experiments showed that after T4-RNA ligase was immobilized, its immobilized phase still kept its essential catalytic property. It was stable and could be used repeatedly as other immobilized enzymes.The preparation procedure-of the immobilized enzyme was simple. It was suitable for the synthesis of nucleic: acid with defined sequences.更多还原