专一切割苹果锈果类病毒多体自切割核酶的克隆和转录物的体外活性测定

Design and Preparation of the Multimeric Self-cleavable Hammerhead Ribozyme Targeting Apple Scar Skid Viroid and Its Activity Detection in vitro

将苹果锈果类病毒的 1个 14nt的靶序列连接在锤头型核酶的 3′末端 ,构成自切割核酶。经人工合成和PCR扩增 ,克隆在转录载体pGEM7zf(+)的XhoⅠ HindⅢ位点。利用限制酶XhoI与SalI的连接 ,消失其识别位点序列 ,将自切割核酶片段插入到重组质粒中 ,经连续 5次亚克隆 ,分别获得 2、4、6、8、10和 12拷贝的多体自切割核酶。在T7RNA聚合酶作用下 ,线性化重组质粒转录的多体自切割核酶通过内部的顺式切割释放出较多数量的核酶分子 ,提示在转录水平能够提高核酶转录物的浓度。用相同摩尔浓度的单体和 12体自切割核酶分别对3 2 P标记的靶ASSVd进行反式切割 ,核酶与靶RNA摩尔浓度比为 1:1。放射自显影结果表明 :多体自切割核酶对靶ASSVd的切割效率明显高于单体自切割核酶。

A self-cleaving hammerhead ribozyme gene containing a 14nt target sequence of ASSVd at the 3′ end of hammerhead ribozyme was synthesized、amplified and cloned at the Xho I-Hind Ⅲ site of pGEM7Zf(+).The ends produced by Xho I or Sal I can link together, thus the recognition sites of both enzymes vanish and can't be cut by either one. We used this property to get the recombinant plasmid bearing 2、4 、6、8、10 and12 copies of self-cleavable ribozyme respectively after successively sub-cloning five times. Linearized recombinat plasmid model catalyzed by T7 RNA polymerase was transcribed in vitro. The multimeric ribozyme molecules efficiently self-cleaved via cis-acting to release many ribozyme molecules It indicates that the concentration of ribozyme transcripts has been enhanced during transcription. Trans-cleavage reaction was carried out by incubating monomeric and multimeric ribozymes with same mol concentration and 32P labeled target ASSVd. Both ribozymes and target transcripts were mixed in 1∶1 ratio. Autoradiograms showed the transcripts of multimeric ribozyme were substantially more effective against the ASSVd target RNA than the monomeric ribozymes. We confer that the multimeric self-clevable ribozyme is likely to provide more valuable application in vivo.