靶向人CD106基因RNAi慢病毒载体的构建与鉴定 |
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引用本文: | 孙乐刚,于婷婷,刘玲,李朝晖,付洪海,王丽芳.靶向人CD106基因RNAi慢病毒载体的构建与鉴定[J].实用口腔医学杂志,2016(6):783-786. |
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作者姓名: | 孙乐刚 于婷婷 刘玲 李朝晖 付洪海 王丽芳 |
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作者单位: | 1. 滨州医学院附属医院口腔外科,256603;2. 滨州医学院附属医院内镜中心,256603;3. 青岛市东南口腔诊所 |
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基金项目: | 山东省自然科学基金(ZR2013HL008),山东省教育厅科技计划项目(J12LK08) |
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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。
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关 键 词: | CD106 RNA干扰 慢病毒载体 |
Construction and characterization of RNAi lentiviral vector targeting human CD106 gene |
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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 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. |
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Keywords: | CD106 RNAi Lentiviral vector |
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