中文摘要：

【目的】构建小鼠Kruppel样因子4RNA干扰慢病毒表达载体，并观察KLF4基因在小鼠腹腔巨噬细胞RAW264．7细胞中的表达情况。【方法】根据GenBank中基因信息，采用干扰序列设计软件设计靶点，合成、制备KLF4-shRNADNA片段，与载体连接构建真核表达慢病毒干扰载体pLVX-KLF4-shRNA，转化感受态，筛选阳性克隆，并进行测序鉴定。在Lipofectamine@3000的介导下，pLVX-KLF4-shRNAl／2与psPAX2、pMD2．G辅助质粒共转染293T细胞，包装产生病毒Lenti-KLF41／2，将重组的慢病毒感染RAW264．7细胞，实时定量PCR法检测KLF4mRNA的表达。收集病毒上清并浓缩测定病毒滴度。【结果】经测序证实，沉默小鼠KLF4基因的真核表达慢病毒干扰载体构建成功，倒置荧光显微镜下可见包装细胞293T细胞表达绿色荧光，RT-PCR证实RAW264．7细胞中KLF4mRNA的敲除效率分别在60％、75％左右（P〈0．05）。初次浓缩后测定小鼠KLF4基因重组慢病毒的滴度为1．65×10^8TU／ml。【结论】成功构建出KLF4的RNA干扰慢病毒载体pLVX-KLF4-shRNA2可用，并包装出慢病毒，为本实验在后续的体内体外研究中探讨KLF4的作用机制奠定了实验基础。

英文摘要：

[Objective]To construct the lentiviral vector for RNA interference （RNAi） of mouse Kruppel-like factor 4 （KLF4） gene and observe the KLF4 expression in mouse RAW264.7 cells. [Methods] According to the data of GeneBank, specific RNAi target sequences were designed and synthesized and KLF4-shRNA DNA sequence was prepared. The RNAi lentiviral vector pLVX-KLF4- shRNA was constructed and transformed into competent cells. Then the sequences were identified. The correct recombinant lentiviral vector pLVX-KLF4-shRNA 1/2 together with the two lentiviral packing vectors psPAX2 and pMD2.G cotransfected the package ceils 293T by Lipofectamine3000. The recombinant lentivirus Lenti-KLF4 1/2 was harvested to infect RAW264.7 cells from 293T cells. The expression of KLF4 mRNA was determined by real-time PCR. The viral supernatant was collected and concentrated for the detection of viral titer. [Results] The sequencing results indicated that the RNAi lentlviral vector pLVX-KLF4-shRNA was constructed successfully. The green fluorescence from 293T cells was observed via inverted fluorescence microscope. The results of RT- PCR showed that the knockout rates of KLF4 mRNA from RAW264.7 cells were about 60% and 75% （P〈0.05）. The titer of mouse recombinant lentivirus of KLF4 （ Lenti-KLF4 2）was 1.65×l0^8 TU/ml after the first concentration. [Conclusion] The construction of RNAi lentiviral vector pLVX-KLF4-shRNA 2 is successful. Therefore, it provides the experimental basis for the following studies invivo and in vitro to explore the mechanism of KLF4.