可表达随机12肽库的逆转录病毒表达系统的建立及评价  

Establishment and Evaluation of Retroviral Expression System Expressing Random 12 Peptide Library

作　　者：赵亚[1] 李英辉[1] 黄豫晓[1] 刘忠湘[1] 雷俊川[1] 李淑梅[1] 刘军[1] 薛采芳[1] 

机构地区：[1]第四军医大学病原生物学教研室,陕西西安710032

出　　处：《生物技术通讯》2008年第6期814-816,共3页Letters in Biotechnology

摘　　要：目的:建立可表达随机12肽库的逆转录病毒表达系统。方法:体外合成编码随机12肽的DNA片段;在最优化的实验参数和反应条件下将DNA片段克隆入带有EGFP标记的逆转录病毒载体后分批次电击转化大肠杆菌,合并转化所得菌液即为可表达随机12肽库的逆转录病毒原始载体库;半固体扩增法扩增该原始载体库,提取质粒并转染GP2-293包装细胞,在EGFP表达最强的时间点收集细胞培养上清,即为可表达随机12肽库的逆转录病毒库。结果:可表达随机12肽库的逆转录病毒原始载体库的库容量为3.14×106cfu;扩增后的逆转录病毒载体库滴度为5.2×109cfu/mL,库容量为2.34×1011cfu;转染了已扩增的载体库质粒后的GP2-293包装细胞可以成功地表达随机12肽库。结论:建立了可表达随机12肽库的逆转录病毒表达系统,为抗病毒寡肽的筛选以及进一步的深入研究奠定了良好的基础。Objective： To establish retroviral expression system expressing random 12 peptide library. Methods： Doublestranded DNA fragments encoding random 12 peptide were synthesized in vitro. The DNA fragments were cloned into retroviral vector with EGFP marker and the ligation products were proceeded to eleetroporation in batch subsequently un- der optimal reaction condition and experiment parameter. All the electroporation products were merged to compose the pri- mary retroviral vector library expressing random 12 peptide. The primary retroviral vector library was amplified by semi- solid amplification and the plasmids of it were transfected into GP2-293 packaging cells. When EGFP expression reach the peak, collect cell culture supematants which composing retroviral library expressing random 12 peptide library. Resuits： Capacity of primary retroviral vector library was 3.14×10^6 cfu; titer of retroviral vector library was 5.2×10^9 cfu/mL and its capacity reached 2.34×10^11 cfu after amplification. GP2-293 packaging cells can successfully express random 12 peptide after being transfected with amplified library plasmids. Conclusion： The retroviral expression system expressing random 12 peptide was established, which may lay a sound foundation for the screening of antiviral oligo-peptide and for further relative research.

关 键 词：肽库 逆转录病毒 电击转化 转染 

分 类 号：Q78[生物学—分子生物学]