细胞外基质金属蛋白酶诱导因子RNAi慢病毒载体的构建

摘要

Objective To construct lentivirus vector for RNA interference( RNAi) targeting extracellular matrix metal-loproteinase inducer(EMMPRIN). Methods RNAi targeting sequence was designed. The complementary Oligo DNA targeting EMMPRIN were synthesized, and then forming double strand DNA after annealing, connecting pGCSIL-GFP vector after enzyme digestion by Age I and EcoR I , forming lentivirus vector pGC-LV. The positive clones containing Emmprin shRNA were identified by PCR amplification and sequencing. 293 T cells were cotransfected with lentiviral vector pGC-LV, pHelper 1.0 and pHelper 2.0 using Lipfectamine 2000. The recombinant lentivirus were collected and detected by dilution method one hole by another. Results A recombinant lentiviral vector which expressed short hairpin RNA targeting Emmprin was successfully constructed and confirmed by DNA sequencing. The recombinant lentivirus was harvested from 293T cells and the viral liter was 1 × 109 TU/ml. Conclusion RNAi lentivirus vector of EMMPRIN was constructed successfully, and the vector provides a basis for further studying of the roles of Emmprin in acute leukemia.%目的 构建人细胞外基质金属蛋白酶诱导因子(EMMPRIN) RNA干扰(RNAi)慢病毒载体.方法 参照文献设计RNAi靶点序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经Age Ⅰ和EcoR Ⅰ酶切后的pGCSIL-GFP载体连接产生RNAi慢病毒载体pGC-LV.PCR鉴定阳性克隆并测序,将测序正确的pGC-LV、pHelper 1.0和pHelper 2.0质粒经脂质体2000共转染293T细胞,获得重组慢病毒,用逐孔稀释法测定病毒滴度.结果 合成的含EMMPRIN vshRNA慢病毒载体寡核苷酸链插入正确；病毒包装后滴度为1×109 TU/ml.结论 应用基因工程技术成功构建了可供感染的EMMPRIN基因RNAi慢病毒载体,此为进一步研究EMMPRIN在急性白血病中的作用奠定了实验基础.