中文摘要：

目的：模拟miR30的框架结构设计针对HPSE的shRNA，将其克隆至慢病毒载体的CMV启动子调控的表达框内，实现Ⅱ型启动子对RNAi的有效调控。方法：设计3条基于miR30骨架的HPSE—shRNA，通过PCR获得目的片段，回收后与线性化OVPP．GFP载体连接产生重组慢病毒载体LVPP—GFP／miR—HPSE—shRNA，PCR筛选阳性克隆，测序鉴定。用LVPP-GFP／miR．HPSE-shRNA、pHelper1．0载体和pHelper2．0载体共转染包装细胞293T细胞，产生慢病毒，感染A375细胞。以实时荧光定量PCR检测HPSE．mRNA的表达情况，以Westenblot检测HPSE蛋白的表达水平。结果：构建出以miR30为骨架的针对HPSE的慢病毒干扰载体，经阳性菌落PCR鉴定与测序，结果正确。实时荧光定量PCR和Westenblot结果表明，LVPP—GFP／miR—HPSE-shRNA重组病毒感染后A375细胞HPSE—mRNA和蛋白的表达明显降低。结论：本研究成功构建了以miR30为骨架的HPSE—shRNA慢病毒载体，实现了Ⅱ型启动子对RNAi的有效调控，为今后实现溶瘤病毒介导RNAi提供了实验依据。

英文摘要：

Objective： To construct a lentiviral RNA interference system targeting heparanase （HPSE） based on miR30 and to test its silencing effect. Methods： Three heparanase-shRNA structures were designed based miR30. The targeting fragments were obtained by PCR,then inserted into the vector LV PP-GFP to construct the recombinant lentiviral vector LV PP-GFP/miR-HPSE-shRNA, which was identified by PCR and sequencing. The 293T cells were co-transfect with LV PP-GFP/miR-HPSE-shRNA, pHelper 1.0 vector and pHelper 2.0 vector to produce lentiviruses,with which A375 cells were infected. Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein. Results： The lentiviral miR30-based RNAi vector targeting heparanase was constructed and confirmed by PCR and sequencing. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of both heparanase mRNA and protein in infected A375 cells were decreased significantly than those in control group. Conclusions： The lentiviral miR30 -based RNAi vector targeting heparanase was been constructed successfully, which can be used for further study on RNAi-mediated oncolytic viruses.