中文摘要：

目的构建能表达CDK5特异性小干扰RNA（siRNA）（cdk5-siRNA）的重组腺相关病毒（AAV）载体，体外观察其对CDK5基因的沉默作用。方法采用基因克隆技术将合成的特异性cdk5RNA干扰寡核苷酸序列克隆至AAV Helper-Free System中的表达质粒pAAVMCS，构建出质粒pAAVM cscdk5-siRNA，通过酶切测序鉴定重组质粒；将该质粒与系统中的控制质粒pAAVRC、辅助质粒pHelper用磷酸钙法共转染HEK293细胞，包装得到表达cdk5-siRNA的重组腺相关病毒载体（rAAV-cdk5-siRNA）；用斑点杂交法测定重组病毒的滴度，重组病毒感染体外培养的PCI2细胞，Western blot检测其抑制cdk5表达的效果。结果成功构建并包装出重组腺相关病毒载体rAAVcdk5-siRNA，病毒滴度达4×10^13／ml，重组病毒感染后的PC12细胞cdk5表达明显下调。结论构建的重组腺相关病毒载体rAAV—cdk5-siRNA能明显干扰cdk5的表达，为将其进一步应用于神经变性疾病的治疗研究奠定了基础。

英文摘要：

Objective To construct the adeno-assoeiated virus vector encoding the small interfering RNA （siRNA） specific for cyclin dependent kinase 5 （edk5） gene and to observe its silencing effect on edk5 gene. Methods The specific cdk5-siRNA sequence was cloned into the plasmid of pAAV-MCS in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-MCS-edk5-siRNA, The recombinant plasmids were identified by DNA sequencing and restriction digestion. Then the packaging cell lines （ HEK293 cell） was cotransfeeted with the pAAV-MCS-cdk5-siRNA together with the control plasmid pAAV RC and pHelper by phosphate-calcium deposit method. The titer was measured by dot hybridization. The recombinant adeno-assoeiated virus vector infected the PC12 cell, at 72 h after infection, the expression of edk5 was detected by Westernblot. Results The recombinant adeno-assoeiated virus vector carrying the edk5 siRNA sequences was construeted successfully. The viral titer was 4 × 10^13 and it down-regulated the expression of the cdk5 gene in PC12 cell. Conclusions The recombinant adeno-assoeiated virus vector rAAV cdk5 siRNA can interfere the expression of cdk5 gene significantly,which lays the basis for its application in the treatment of the neurodegenerative disease.