靶向EBV潜伏期基因EBNA2小干涉RNA系统的构建  

Construction of Small Interfering RNA System Targeting EBV Latent Gene EBNA2

作　　者：孙淑红[1] 胡晓峰[2] 王笑峰[1] 殷凡[3] 罗兵[1] 

机构地区：[1]青岛大学医学院微生物教研室,山东青岛266021 [2]山东省临沂市人民医院,山东临沂276003 [3]青岛市疾病控制中心,山东青岛266033

出　　处：《肿瘤学杂志》2007年第1期40-43,共4页Journal of Chinese Oncology

基　　金：国家自然科学基金资助项目(30471623)

摘　　要：[目的]构建携带靶向EBV核抗原EBNA2(EBV nuclear antigen,EBNA)的pSUPER.retroRNAi逆转录病毒载体,进而筛选出稳定产毒的细胞克隆。[方法]用DNA重组技术将60nt能转录产生靶向EBNA2小发夹RNA(smallhairpin RNA,shRNA)的寡核苷酸序列定向插入逆转录病毒载体pSUPER.retro,并用限制性内切酶酶切和测序鉴定;脂质体法将重组逆转录病毒载体转染包装细胞系PheonixA,G418筛选稳定产生逆转录病毒的细胞克隆。[结果]重组逆转病毒载体经限制性内切酶酶切,电泳后可观察到7167bp和281bp两条DNA条带;测序鉴定结果表明序列正确;重组载体转染的包装细胞经G418筛选获得能产生逆转录病毒的抗性细胞克隆,病毒滴度为2.5×104CFU/ml。[结论]成功构建和筛选出靶向EBV潜伏期基因EBNA2的pSUPER retroRNAi逆转录病毒载体以及稳定产毒的细胞系,为深入探讨EBNA2基因的细胞转化和抗凋亡作用提供了实验基础。[Purpose ] To construct a recombinant retroviral vector pSUPER-EBNA2 that targets EBV nuclear antigen 2 （EBNA2） and then to sift a stable virus-producing cell line. [Methods] The 60nt nucleiotide sequence encoded targeting EBNA2 shRNA was cloned into a retroviral vector pSUPER, retro with DNA recombinant technique, and the recombinant vector was confirmed by the restrictive enzyme analysis and DNA sequencing. The packaging cell Pheonix A was transfected with recombinant plasmid using liposome-based transfection method and the stable intergrant was selected by using G-418. [ Results ] The restrictive enzyme analysis and electrophoresis showed two DNA strips which were 7167bp and 281bp, respectively. The result of DNA sequencing demonstrated that the insertion sequence was exactly correct. The recombinant vector was transfected into the packaging cell, and then the anti-G418 positive clones which can excrete recombinant retrovirus were sieved out. The tite of the recombinant retrovirus is 2.5×10^4 CFU/ml. [Conclusion] A recombinant retroviral vector pSUPER-EBNA2 and a stable virus-producing cell line were constructed and sifted successfully which provide on experimenteal base to study the effect of cell transformation and anti-apoptosis in EBNA2 gene.

关 键 词：小发夹RNA 核抗原EBNA2 Pheonix A 逆转录病毒载体 EB病毒 

分 类 号：R373[医药卫生—病原生物学]