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目的:进一步研究整合素连接激酶(ILK)的功能,构建ILK基因RNAi慢病毒载体,并对其在肺腺癌A549细胞株中干扰效率进行鉴定。方法:针对ILK基因RNAi有效靶序列,合成4对oligo DNA,退火形成双链DNA,与经AgeI和EcoRI酶切的质粒pGCSIL-GFP载体连接产生shILK-LV慢病毒载体,PCR筛选阳性克隆,测序鉴定,用shILKi-LV载体、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞绿色荧光蛋白(GFP)的表达水平测定病毒滴度。获得重组慢病毒后感染人肺癌A549细胞,荧光显微镜观察GFP及Real-time PCR检测ILK在A549细胞中的表达。结果:PCR扩增出插入片段,测序证实成功构建了ILK-shRNA慢病毒载体shILKi-LV。包装并浓缩慢病毒,滴度分别为6×108、6×108、5×108和4×108 pfu/mL;将病毒感染A549细胞,荧光显微镜下观察80%细胞表达绿色荧光,Real-time PCR检测ILK mRNA表达明显下降,其中染shILKi-KD-1的A549细胞中ILK2-ΔΔCt值为0.058,shILKi-KD-2、3和4的ILK2-ΔΔCt值分别为0.162、0.072和0.119,尤以ILKsiRNA-1抑制最明显。结论:成功构建shILKi-LV病毒载体并建立了A549-shILKi细胞模型,为ILK在肺癌信号转导通路中的研究提供了工作基础。
OBJECTIVE: To further investigate the function of ILK and to construct RNAi lentivirus vector of ILK gene, and to identify its interference efficiency in lung adenocarcinoma A549 cell line. METHODS: Four pairs of oligo DNA were synthesized and annealed to form double-stranded DNA. The recombinant plasmid was cloned into plasmid pGCSIL-GFP vector digested with AgeI and EcoRI to generate shILK-LV lentiviral vector. The positive clones were screened by PCR and sequenced The 293T cells were co-transfected with the shILKi-LV vector, pHelper 1.0 vector and pHelper 2.0 vector. The lentivirus was packaged and the virus titer was determined by the expression level of green fluorescent protein (GFP) in 293T cells. Human lung adenocarcinoma A549 cells were infected with the recombinant lentivirus. The expression of ILK in A549 cells was detected by fluorescence microscopy and Real-time PCR. Results: The inserted fragment was amplified by PCR. Sequencing confirmed that ILK-shRNA lentivirus vector shILKi-LV was successfully constructed. The lentiviruses were packaged and concentrated with the titer of 6 × 108, 6 × 108, 5 × 108 and 4 × 108 pfu / mL, respectively. A549 cells were infected with virus and 80% of the cells were observed under fluorescent microscope. Real-time PCR The ILK2-ΔΔCt value of A549 cells transfected with shILKi-KD-1 was 0.058, ILK2-ΔΔCt values of shILKi-KD-2, 3 and 4 were 0.162, 0.072 and 0.119, respectively, especially ILKsiRNA -1 inhibition is the most obvious. Conclusion: The shILKi-LV virus vector was successfully constructed and a A549-shILKi cell model was established, which provided a basis for the study of ILK in lung cancer signal transduction pathway.