重组腺病毒介导RNA干扰人DLK1基因表达的研究

RNA Interference with Human DLK1 Gene Mediated by Recombinant Adenovirus

构建抑制人DLK1基因表达的重组腺病毒载体,利用腺病毒载体介导的RNA干扰技术评价其在肝癌细胞株中的基因沉默效应.将针对人DLK1基因的RNAi寡核苷酸序列,连接到腺病毒穿梭质粒中,在含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183内进行同源重组.重组腺病毒载体在HEK-293细胞中包装扩增,得到高滴度的重组腺病毒.通过绿色荧光蛋白示踪腺病毒的感染效果,并通过荧光实时RT-PCR,Western blot的方法证实重组腺病毒能够显著抑制DLK1基因在肝癌细胞株中的表达.

High-effective vectors for knockdown of DLK1 gene were constructed by adenovirus-mediated RNAi technology and the RNA interference effect of DLK1 was evaluated in human hepatocellular carcinoma （HCC） cell lines. The oligonucleotide sequences encoding the DLKl-specific siRNA cassettes were cloned into the AdEasy shuttle vector pShuttle and homogonously recombined with an adenoviral backbone plasmid pAdEasy-1 in competent E. coli BJ5183 cells. The resultant adenoviral plasmids were packed in HEK293 cells, and virus stocks were propagated and obtained high-titer infective adenoviruses. The effect of adenovirus infection to HCC was confirmed by tracing with GFP. With the results of real time RT-PCR and Western blot, the adenoviruses could significantly decrease DLK1 gene expression in HCC cell lines.