靶向人CD106基因RNAi慢病毒载体的构建与鉴定

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1×109 TU/ 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。%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 lentivi-ral expression vector. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids were trans-fected into 293T cells to harvest siRNA lentivirus. After infection in HN12 cells, Real-time PCR and western blot were performed to de-termine 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 sig-nifically, 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.