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。
Objective： To construct CD106-targeted RNAi lentiviral vector plasmids. Methods： 4 targets aimed at C D106 （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.
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2016年第6期783-786,共4页
Journal of Practical Stomatology
基金
山东省自然科学基金(编号:ZR2013HL008)
山东省教育厅科技计划项目(编号:J12LK08)
