| 慢病毒Lenti-CXCR4-siRNA载体的构建 |
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| 引用本文： | 王天宝,林维浩,石汉平,韩方海,董文广.慢病毒Lenti-CXCR4-siRNA载体的构建[J].中华全科医学,2012,10(10):1496-1498,1661. |
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| 作者姓名： | 王天宝 林维浩 石汉平 韩方海 董文广 |
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| 作者单位： | 中山大学附属第一医院外科 |
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| 基金项目： | 广东省科技计划资助项目(2010B060900100) |
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| 摘 要： | 目的构建介导CXCR4 RNA干扰的Lenti-CXCR4-siRNA慢病毒载体。方法将人工合成的CXCR4 siRNA经PCR扩增聚合后与线化的载体GV115连接,XhoI酶切及测序鉴定该表达质粒pLenti-CXCR4-siRNA,按Lenti-X Bicis-tronic Expression System操作手册构建Lenti-CXCR4-siRNA慢病毒,纯化后测定其滴度。结果凝胶电泳显示PCR扩增聚合产物大小正确(约60 bp)。表达质粒pLenti-CXCR4-siRNA可被XhoI内切酶线化。碱基测序证实碱基序列与设计序列一致。重组Lenti-CXCR4-siRNA慢病毒的包装细胞293T可见绿色荧光,纯化后测定其滴度,病毒滴度为2×109TU/ml。结论成功构建了高滴度的重组Lenti-CXCR4-siRNA慢病毒。
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| 关 键 词： | CXCR4 RNA干扰 慢病毒 293T细胞 |
The Construction and Purification of Lentivirus Lenti-CXCR4-siRNA Mediating CXCR4 RNA Interference |
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| Institution: | WANG Tian-bao,LIN Wei-hao,SHI Han-ping,et al.Surgery Department,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,Guangdong,China |
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| Abstract: | Objective To construct Lenti-CXCR4-siRNA lentivirus which mediates CXCR4 RNA interference(RNAi).Methods The CXCR4 RNA interference sequence ligated with GV115,and formed a new plasmid named pLenti-CXCR4-siRNA.Lenti-CXCR4-siRNA lentivirus was constructed following the manual of operations of Lenti-X Bicistronic Expression System,and virus titer was calculated after purification.Results Gel electrophoresis showed that the size of PCR product was about 60 bp.The expression plasmid pLenti-CXCR4-siRNA could be digested by Xho I incision enzyme.Gene sequencing verified the insertion sequence was correct.Lenti-CXCR4-siRNA could make 293T cells present green fluorescence,and its titer was 2×109 TU/ml after purification.Conclusion A high titer Lenti-CXCR4-siRNA was reconstructed correctly. |
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| Keywords: | CXCR4 RNA interference Lentivirus 293T cell |
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