慢性粒细胞白血病特异性单、双及三单位核酶载体的构建

Construction and identification of single ,double and triple unit ribozymes for treatment of chronic myeloid leukemia

本文拟针对在慢性粒细胞白血病发病中起关键作用的ｂｃｒａｂｌ 融合基因， 构建特异性的系列核酶载体。为此，我们针对ｂｃｒａｂｌ 融合位点设计、合成了３ 个相邻的锤头状核酶。通过基因重组将３ 个单核酶按顺序定向克隆入改建的ｐＧＥＭ３ｚｆ 载体中，构建了ｂｃｒａｂｌ 单核酶、双核酶及三核酶体外转录载体， 并在此基础上将三单位核酶克隆入ｐ ＤｏＲｎｅｏ 载体中构建三核酶逆转录病毒载体。此外， 通过ＰＣＲ 方法， 扩增了ｂｃｒａｂｌ 融合位点附近约３７６ｂｐ 的序列，成功地构建了ｂｃｒａｂｌ 融合基因体外转录载体。本文构建的ｂｃｒａｂｌ 特异性系列载体，为今后遴选特异、高效的核酶打下了基础，并为将核酶用于自体骨髓移植的体外骨髓净化创造了条件。

Bcr abl fusion gene plays an important role in mechanism of chronic myeloid leukemia. To investigate the ribozyme's potential application in the treatment of CML, series vectors containing bcr abl specific ribozymes were designed and constructed in this paper. Firstly, three hammer head ribozymes specific to the fusion point of bcr abl were designed and cloned into a modified in vitro transcription vector pDES. After recombination, a group of single , double and triple unit ribozyme in vitro transcription vectors and a triple unit ribozyme retroviral vector were constructed. The successful construction of recombinant vector was identified by DNA sequencing. Secondary, a 376bp fragment containing the bcr abl fusion site was amplified by PCR. This fragment was cloned into pBluescript KS to be used as the template vector for in vitro cleavage experiment. The construction of series of bcr abl specific ribozyme vectors made a solid foundation for further specificity and efficiency identification of different bcr abl ribozymes. It was also the basement for using ribozyme in auto bone marrow transplantation.