抗慢性粒细胞性白血病bcr/abl mRNA真核表达载体的构建

目的：构建一个含M1RNA、具有特异性抗慢性粒细胞性白血病（CML）细胞融合基因bcr/ablmRNA的真核表达载体，以用于CML的分子靶向治疗。方法：以pTK117为模板，通过PCR方法合成一个带有导引序列（GS）的M1RNA，再将PCR产物克隆到真核表达载体pNAV-1上，得到重组质粒pAVGS4，转化大肠埃希菌JM109，以碱裂解法小量抽提，酶切电泳鉴定，并测序鉴定。结果：以EcoRI和SalI酶切、1％的琼脂糖凝胶电泳显示在500和6500bp附近各有一条明亮的条带。应用PCR方法测序的结果与模板序列一致。结论：构建的pNAV-1经酶切和测序鉴定，与目标序列一致，含有核酶、具有抗bcr/abl mRNA的真核表达载体构建成功，预期可用于CML细胞株和原代细胞的实验研究。

Construction of Eukaryotic Expression Vector Against bcr/abl mRNA in Chronic Myelogenous Leukemia Cell

Purpose To construct a eukaryotic expression vector with M1-GS RNA as a molecular target tool to cleave bcr-abl mRNA in chronic myelogenous leukemia cell. Methods pTK117 was used as PCR a template to amplify M1 RNA with guide sequence (GS). After M1?GS RNA being subcloned to pNAV?1, pAVGS4 was constructed. The recombination plasmid was amplified by transforming to JM109. Restriction enzyme analysis and DNA sequencing were used to identify the recombinant plasmid. Results pAVGS4 was determined by restriction enzyme analysis? One percent agarose electrophoresis showed that there are two bands near 500 and 6 500 bp respectively. Then pAVGS4 was sequenced by PCR. The sequencing result showed that pAVGS4 contained M1?GS RNA sequence. Conclusions The eukaryotic expression vector containing ribozyme against bcr/abl mRNA has been successfully conducted.