摘要
目的构建并筛选携带针对Epstein-Barr病毒(EBV)潜伏膜蛋白基因LMP1的pSUPER retroRNAi逆转录病毒载体以及稳定产毒的细胞克隆。方法用DNA重组技术,将60 bp能转录产生靶向LMP1小发夹RNA(shRNA)的寡核苷酸序列,定向克隆入逆转录病毒载体pSUPER retro,脂质体法将重组逆转录病毒载体转染包装细胞系PA317,G418筛选建立稳定产生逆转录病毒的细胞克隆。结果重组逆转录病毒载体经限制性内切酶酶切,电泳后可观察到7 167 bp和281 bp两条DNA条带;测序鉴定结果表明序列正确;重组载体转染包装细胞,可表达绿色荧光蛋白,经G418筛选,得到抗性细胞克隆。结论特异性沉默EBV潜伏期基因LMP1的pSUPER retro RNAi逆转录病毒载体以及稳定产毒的细胞系构建和筛选成功。
Objective To construct and identify a recombinant retroviral vector pSUPER-LMP1 that target EBV latent membrane protein gene 1 （LMP1） and a stable virus-producing cell line. Methods The 60 bp encoded targeting LMP1 gene shRNA sequence was cloned into a retroviral vector pSUPER retro with DNA recombinant technique. The recombinant vector was identified by the electrophoresis analysis of restriction enzyme digestion and DNA sequencing. The packaging cell PA317 was transfected with this recombinant plasmid using liposome-based transfection and the stable intergrant was selected by using G418 medium. Results The electrophoresis of EcoR Ⅰ and HindⅢ digested products showed two DNA fragments, 7 167 bp and 281 bp, respectively. The result of sequence demonstrated that 60 bp had been inserted into the vector. Green fluorescent protein （GFP） was observed after transfection. G418 resistant clones were also selected. Conclusion A recombinant retroviral vector pSUPER-LMP1 and a stable virus producing packaging cell line were successfully constructed and identified.
出处
《齐鲁医学杂志》
2006年第3期197-200,共4页
Medical Journal of Qilu
基金
国家自然科学基金资助项目(30471623)
