【摘要】 目的:构建人CD106基因RNAi慢病毒载体。方法:设计4条靶向CD106的RNA干扰靶点序列(Target 1、2、3、4),合成短发卡结构shRNA并退火成双链DNA,与慢病毒载体重组形成siRNA表达载体,利用PCR和测序鉴定验证获得连接正确的克隆。经由293T细胞包装siRNA慢病毒颗粒,随后将其感染人口腔鳞癌HN12细胞,分别采用Real-time PCR和Western blot方法检测靶基因在mRNA和蛋白质水平的沉默效率。结果:构建的慢病毒载体的PCR鉴定和测序正确,包装病毒后滴度至少达到1×109TU/ml。siRNA慢病毒感染人HN12细胞,经Real-time PCR和Western blot检测目的基因CD106的mRNA和蛋白表达较阴性对照载体慢病毒感染组明显下降。结论:成功构建了人靶向CD106RNAi慢病毒载体,并能够在细胞水平上有效沉默靶基因。更多还原

【Abstract】 Objective: To construct CD106-targeted RNAi lentiviral vector plasmids. Methods: 4 targets aimed at CD106( Target 1,2,3,4) were designed. Oligo-DNA fragment containing short hairpin frame was synthesized and reannealed,and then cloned into lentiviral expression vector. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids were transfected into 293 T cells to harvest siRNA lentivirus. After infection in HN12 cells,Real-time PCR and western blot were performed to determine the expressing level of CD106. Results: PCR and sequencing revealed that siRNA plasmids was correctly constructed. Virus with a titer of 1 × 109 TU / ml was successfully packaged at least. CD106 expression in HN12 cells could be knockdown by virus infection significally,compared with negative control lentivirus. Conclusion: The recombinant lentiviral siRNA expressing vector targeting human CD106 gene has been successfully constructed and packaged. CD106 gene in cells may be down-regulated by lentiviral siRNA.更多还原