人PAX6基因shRNA慢病毒载体的构建及鉴定

Construction of the human PAX6 gene shRNA lentiviral vector and evaluation of RNAi efficiency

目的体外构建人PAX6基因的shRNA慢病毒载体并鉴定。方法实验研究。设计PAX6基因特异性siRNA靶点,构建PAX6 shRNA重组质粒,构建表达FLAG tag、PAX6以及红色荧光蛋白报告基因的融合蛋白表达质粒,经Western Blot加以验证,使用293T细胞进行有效靶点的外源筛选。构建慢病毒pGCL-GFP载体,在293T细胞中包装病毒,将shRNA重组病毒感染人晶状体上皮细胞系(HLE-B3),检测B3细胞PAX6蛋白表达情况,从而筛选有效靶点。根据PAX6基因的4个候选RNAi靶序列,构建shRNA重组质粒,并构建FLAG tag、PAX6、红色荧光蛋白融合蛋白的表达系统。结果经验证外源PAX6基因,发现3个候选靶点是有效的RNAi靶点,能使PAX6蛋白表达水平显著降低。在此基础上构建的PAX6 shRNA重组慢病毒载体,显著抑制了HLE-B3细胞PAX6蛋白的内源性表达。结论成功构建了人PAX6基因的shRNA慢病毒表达载体,在分子水平能够有效沉默靶基因,为后续探讨PAX6对晶状体上皮细胞的生物学作用奠定了基础。

Objective To construct the human PAX6 gene shRNA lentiviral vector and identify the interference efficiency in the HLE-B3 human lens epithelial cell line. Methods It was an experimental study. PAX6 gene-specific siRNA targets were designed. PAX6 shRNA recombinant plasmid vector,fusion protein expression plasmid of FLAG tag,PAX6 and a red fluorescent protein reporter gene were built and then were verified by Western Blot. Exogenously screened was taken to find effective target with 293T cells. Packaged virus with the three-plasmid system to infect HLE-B3 cells,PAX6 protein expression to find effective target. Constructed shRNA recombinant plasmids respectively in accordance with the four candidate PAX6 gene RNAi target sequences. Constructed the Flag-tag,PAX6 as well as red fluorescent protein fusion protein expression system. Results It was proved that the three candidate target were effective RNAi targets which enabled PAX6 protein decreased significantly. And the three PAX6 shRNA recombinant lentiviral vector which were constructed on this basis could inhibit B3 human lens epithelial cell line from expressing endogenous PAX6. Conclusions Constructed the PAX6 gene shRNA lentiviral expression vector and silenced the target genes at the molecular level effectively,which laid the foundation to explore the biological role of PAX6 in the lens epithelial cells.