针对bcr-abl融合基因不同位点的三个单核酶体外切割活性的比较

Study on the cleavage ability of three ribozymes specific to the different sites of bcrabl fusion gene

目的：针对ｂｃｒ－ａｂｌ融合基因设计并构建３个针对不同位点的特异性单核酶体外转录载体，通过体外切割试验比较其对ｂｃｒ－ａｂｌ融合基因的切割效率，为慢粒基因治疗打下基础．方法：①首先针对ｂｃｒ－ａｂｌ融合位点设计、合成３个相邻的锤头状核酶，将其定向克隆入改建的ｐＤＥＳ载体中，构建３个单核酶体外转录载体并进行序列测定．②同时通过ＰＣＲ方法扩增ｂｃｒ－ａｂｌ融合位点３７６ｂｐ序列，克隆入ｐＢｌｕｅｓｃｒｉｐｔＫＳ载体中构建融合基因体外转录载体，作为核酶体外作用的模板．③分别将核酶及模板进行体外转录及同位素标记，通过切割试验比较不同核酶的体外切割活性．结果：本文通过体外切割试验证实３个单核酶对ｂｃｒ－ａｂｌ融合基因的切割效率分别为５４．６％，２５．３％及３．６％，核酶１效率最高．结论：针对ｂｃｒ－ａｂｌ融合基因不同位点的核酶具有不同的切割效率．本试验结果为遴选、研制高效的ｂｃｒ－ａｂｌ特异性单核酶及多核酶，进一步开展慢性粒细胞白血病核酶基因治疗创造了条件．

AIM: P210 protein encoded by bcrabl fusion gene plays an important role in the mechanism of chronic myeloid leukemia. In order to investigate the potential application of ribozymes in the treatment of CML, three bcrabl ribozymes specific to the fusion site were designed and their cleavage abilities were compared in this experiment. METHODS: Three hammerhead ribozymes specific to the fusion point of bcrabl were designed and successfully cloned into a modified in vitro transcription vector pDES identified by DNA sequencing. A 376 bp fragment containing the bcrabl fusion site was amplified by PCR, which contained the cleavage sites of these 3 ribozymes. This fragment was cloned into pBluescript KS and used as the template vector for in vitro cleavage experiment. After in vitro transcription of template vector and 3 ribozyme vectors, the cleavage abilities of these 3 ribozymes were compared by their in vitro cleavage reaction. WTHZRESULTS: In this experiment, 3 bcrabl specific ribozyme vectors and template vector were successfully constructed. By in vitro cleavage experiment, the cleavage efficiencies of these three ribozymes were identified as 54.6%, 25.3% and 3.6% respectively. Ribozyme 1 was the most effective among them. ZCONCLUSION: Ribozymes to bcrabl fusion gene could specifically digest this fusion gene with different efficiency. This result laid a solid foundation for further study of double and triple unit ribozymes to bcrabl.