一种基于人 miR -30结构高效干扰 LMP1基因表达的慢病毒载体的构建.pdfVIP

现代肿瘤医学 2014年8月 第22卷第8期 MODERN ONCOLOGY，Aug.2014，VOL.22，NO.08 ·1739 · ?论 著? ?基础研究? 一种基于人miR-30 结构高效干扰LMP1基因表达的慢病毒载体的构建 1 2 1 1 1 3 黄必军 ，杨长福 ，朱颖慧 ，周 玲 ，朱秀云 ，黄铁军 Ｃｏｎｓｔｒｕｃｔｉｏｎ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｔｈｅ ｌｅｎｔｉｖｉｒａｌ ｖｅｃｔｏｒ ｃａｒｒｙｉｎｇ ａ ｍｉＲ -30ａ -ｂａｓｅｄ ｓｈＲＮＡ ｔａｒｇｅｔｉｎｇ ＥＢＶ ＬＭＰ1ｏｎｃｏｇｅｎｅ 1 2 1 1 1 3 Huang Bijun ，Yang Changfu ，Zhu Yinghui ，Zhou Ling ，Zhu Xiuyun ，Huang Tiejun 1State Key Laboratory of Oncology in South China，Sun Yat - Sen University Cancer Center，Guangdong Guangzhou 10060，Chi- 2 3 na；De artment of Cancer Chemothera y，the Peo les Hos ital of Gaozhou，Guangdong Gaozhou 2 200，China；De artment of Nuclear Medicine，the Second Peo les Hos ital of Shenzhen，Guangdong Shenzhen 11300，China. 【Abstract】 Objective：To investigate the feasibility and validity of the construction of a lentiviral vector carrying shRNA against EBV LMP1 oncogene.Methods：Based on miR-30a sequence，four LMP1-targeted RNAi shRNA candidate sequenceswere designed and cloned alone with EGFR reporter gene as a cocistron.The plasmid carrying LMP1shRNAandtheLMP1-expressingplasmidwerecotransfectedinto239FTcells，andscreenedthemosteffective shRNA targeting LMP1and constructed lentiviral vector and characterized.Results：The most effective shRNA silen- cing viral LMP1 oncogene was screened successfully and more than 9 % LMP1 protein was knocked down using Western blotting analysis.In 20ml 239FT cell culture supernatant 8 ，about 7.2 ×10 viable particles of recombinant 9 lentiviruswas packaged and the t