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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。
Objective To construct Lenti-CXCR4-siRNA lentiviral vector mediated by CXCR4 RNA interference. Methods The recombinant plasmid pLenti-CXCR4-siRNA was constructed by ligating the synthetic CXCR4 siRNA to the linearized vector GV115 after PCR amplification. The Lenti-X Bicis- CXCR4-siRNA lentivirus and its titer was determined after purification. Results The results of gel electrophoresis showed that the size of PCR product was correct (about 60 bp). The expression plasmid pLenti-CXCR4-siRNA can be XhoI endonuclease linearized. Base sequencing confirmed that the base sequence was consistent with the designed sequence. The recombinant Lenti-CXCR4-siRNA lentivirus packaging cells 293T visible green fluorescence, purified to determine its titer, the virus titer of 2 × 109TU / ml. Conclusion High titer recombinant Lenti-CXCR4-siRNA lentivirus was successfully constructed.