中文摘要：

目的构建大鼠整合素连接激酶-RNAi（ILK-RNAi）慢病毒表达载体,为下一步干预研究细胞转分化及其信号通路奠定基础。方法针对大鼠ILK基因RNAi有效靶序列,合成4条干扰（KD）序列。与pGCSIL-GFP载体连接产生shILK-RNAi慢病毒载体。将连接产物转入制备完毕的细菌感受态细胞,PCR鉴定阳性克隆,测序验证。包装慢病毒,以绿色荧光蛋白（GFP）表达水平验证病毒滴度。慢病毒载体感染大鼠NRK-52E细胞后,RTPCR、WB检测及荧光显微镜观察ILK在其细胞中的表达。结果测序验证成功构建3条大鼠ILK-RNAi慢病毒载体,滴度分别为5×10^8,2.5×10^8,3.5×10^8 pfu/mL。分别以不同感染复数（MOI）感染NRK-52E细胞进行内源筛靶,荧光观察80%细胞的绿色荧光蛋白,RT-PCR、WB检测ILK的表达明显下降。相对于阴性对照序列,KD2,KD3在高MOI组中对ILK表达的敲减达到65%以上（P〈0.05）,为有效靶点。其中KD2及KD3的ILK 2-△△Ct值分别为0.337,0.217。KD2序列为对ILK的表达抑制最显著。结论成功构建大鼠ILK-RNAi慢病毒表达载体。

英文摘要：

Objective To lay the basis for the following study on epithelial-mesenchymal transition and signal pathway through construction of RNA interference （RNAi） lentiviral vector targeting rat integrin-linked kinase （ILK）gene. Methods The complementary oligo DNA containing target sequence was designed, synthesized and cloned into the pGCSIL-GFP vector according to the effective sequence of small interfering （siRNA） targeting ILK gene. The obtained lentiviral vector which expressed short hairpin RNA containing ILK was confirmed by PCR and sequencing. All virus stocks were produced and then transfect into NRK-52E cells in vivo, the titer virus was tested according to the expression level of GFP. ILK expression was assayed by Real-time PCR and Western blotting. Re- sults ILK interference vector was successfully constructed. The titer virus with was 5 ×10^8 , 2.5 ×10^8 , 3.5 ×10^8 pfu/mL. ILK expression in NRK-52E cells could be knockdown by virus infection as characterized by more than 65% decrease of ILK mRNA. Compared with negative control lentivirus, the value expression of KD-2 and KD-3 were 0. 337 and 0. 217 respectively, the function of ILKsiRNA--2 was significant. Conclusion The recombinant lenti- viral shRNA expressing vector targeting rat ILK gene has been successfully constructed and packaged.