中文摘要：

目的：构建针对膜联蛋白（annexin）Ⅱ的短发夹RNA（shRNA）的表达载体，观察其对目的基因表达的影响。方法：根据GenBank中annexinⅡ基因已知序列设计了2条序列，即annexinⅡ shRNA1、annexinⅡ shRNA2，同时设计1条非特异性序列作为阴性对照。据此设计合成各自的寡核苷酸链，退火后连接人pGenesill载体，转化扩增后进行序列测定。3种重组表达载体均转染HEK293细胞，通过实时荧光定量RT—PCR和Western印迹法分别在基因和蛋白水平上检测各自annexinⅡ的表达，以未转染细胞作为空白对照。结果：3种重组表达载体（pGenesillannexinⅡ shRNAl、pGenesill—annexin ⅡshRNA2和pGenesill—negativesh RNA）经限制性酶切及测序分析证明基因插入正确。转染HEK293细胞后，转染pGenesill—annexin ⅡshRNA2组细胞annexinⅡ基因和蛋白表达明显低于转染pGenesill—annexin ⅡshRNAl、pGenesill—negativesh RNA以及空转染细胞（P〈0．05），而后三者间没有明显差异。结论：成功构建了针对annexin Ⅱ的shRNA表达载体（pGenesill—annexin ⅡshR—NA2），转染细胞后可抑制annexinⅡ基因表达。

英文摘要：

Objective：To construct a vector encoding short hairpin RNA（shRNA） targeting annexin Ⅱ and to observe its influence on annexinⅡ expression. Methods： Annexin Ⅱ-targeted hairpin RNA1 and RNA2 were devised according to the GenBank annexin Ⅱ sequence. Meanwhile, a nonspecific sequence was taken as negative control. The oligonucleotide strands of DNA fragments encoding the above shRNAs were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into human pGenesill plasmid followed by amplification in E. coli and sequencing. The 3 pGenesill constructs were then transfected into HEK293 cells and the expression of annexin Ⅱ was examined by real-time fluorescence quantitative RT-PCR and Western bolt. The non-transfected cells were taken as blank control. Results： Restrictive enzyme digestion and sequencing verified that the 3 recombinants（pGenesill-annexin Ⅱ shRNA1, pGenesill-annexin Ⅱ shRNA2, and pGenesill- negative shRNA） were correct. The annexin Ⅱ expression was significantly lower in HEK293 cells transfected with pGenesill-annexin Ⅱ shRNA2 compared with those transfected with pGenesill annexin Ⅱ shRNA1, pGenesill-negative shRNA, and blank control （P〈0.05） ; and there was no significant difference between the latter 3. Conclusion： We have successfully generated an annexin Ⅱ-targeted shRNA construct, which can significantly inhibit the expression of annexin Ⅱ.