稳定表达人SidT2基因细胞系的建立及其dsRNA转运功能的研究

Establishment of Cell Lines Stably Expressing Human SidT2 Gene and Their dsRNA Transportation Function

目的:构建稳定表达人SidT2基因的BHK及MDCK细胞系,探讨SidT2基因过表达与细胞转运双链RNA(dsRNA)能力的关系。方法:根据人SidT2基因序列设计引物,克隆其编码区序列,经双酶切后与pEGFP-N3载体连接,构建其真核表达载体,分别瞬时转染BHK及MDCK细胞,并使用G418筛选稳定表达细胞系;在此基础上,体外转录合成绿色荧光蛋白(GFP)dsRNA,以GFP基因为报告基因,进一步分析过表达人SidT2基因对BHK及MDCK细胞转运dsRNA能力的影响。结果:经基因克隆、酶切、连接后,构建了人SidT2基因真核表达载体pEGFP-SidT2;经瞬时转染及G418筛选,获得稳定过表达人SidT2基因的BHK及MDCK细胞系,实时荧光定量RT-PCR分析表明,其SidT2基因转录水平分别提高71、64.5倍;稳定表达SidT2基因后,在培养液中添加GFP dsRNA,GFP荧光强度较对照细胞分别降低88.1%、73.7%,表明稳定表达SidT2基因的BHK、MDCK细胞转运dsRNA的能力显著增强。结论:构建了稳定表达人SidT2基因的BHK及MDCK细胞系,SidT2基因过表达可显著提高外源性dsRNA的转运能力。

Objective： To establish BHK and MDCK cell lines stably expressing human SidT2 gene, then analyze the relationship between SidT2 gene over expression and cell ability of autonomous transportation of dsRNA. Meth-ods： Primers were designed according to the human SidT2 gene in GenBank, then the ORF of human SidT2 gene were amplified and cloned into pEGFP-N3 vector, BHK and MDCK cells were transfected with recombinant vec- tors and negative control pEGFP-N3 by using LipofectAMINE 2000 reagent, respectively. Following selection with G418, the stable SidT2 over expressing cells and the pEGFP-N3-tranfected BHK and MDCK control cells in form of single colony were screened out. SidT2 expression in the stably transfected cell lines was identified by real time quantitative RT-PCR. The GFP-dsRNA silencing efficiency in SidT2 stably transfected cell lines, and BHK and MDCK control cells were detected by using fluorescence microscope and flow cytometry. Results： The vector pEGFP-SidT2 was constructed by inserting the ORF of human SidT2 gene into the pEGFP-N3 vector. Real time quantitative RT-PCR showed that human SidT2 gene expressed in SidT2 stable transfected BHK and MDCK cell lines were 71 and 64.5 folds higher than respective control cells. The fluorescence of reporter GFP decreased by 88.1% and 73.7% in the SidT2 stable transfected BHK and MDCK cells, indicating that these cell lines can up- take dsRNA from medium more efficiently. Conclusion： BHK and MDCK cell lines stable expression of human SidT2 gene were constructed, and over expression of SidT2 gene can significantly improve the function of cell transportation of dsRNA.