干扰AFP蛋白表达的shRNA表达载体的构建及活性鉴定  

Construction and Activity Detection of Recombinant Plasmid with AFP-Targeted shRNAs

作　　者：张国英[1] 刘明社[1] 赵中夫[1] 张芸[1] 杨慧[2] 武延隽[1] 

机构地区：[1]长治医学院肝病研究所,046000 [2]山西医科大学

出　　处：《长治医学院学报》2007年第5期324-327,共4页Journal of Changzhi Medical College

基　　金：山西省教育厅技术开发基金项目(200611036);山西省自然科学基金项目(20051114)

摘　　要：目的:利用含报告基因EGFP的pGenesil-1质粒构建干扰AFP蛋白表达的短发夹RNA (short hairpin RNA,shRNA)表达的载体,并进行活性鉴定。方法:设计表达短发夹RNA的互补DNA序列,经退火成双链,克隆至带有U6启动子的质粒载体pGenesil-1中,构建重组质粒,转化大肠杆菌DH5α菌株,扩增,提取质粒,酶切鉴定后测序分析。构建正确的重组质粒经脂质体转染HepG2细胞,检测转染率和培养细胞上清的AFP含量变化,确定重组质粒的活性。结果:构建了靶向AFP蛋白的2个shRNA重组质粒载体pGenesil-1-siAFP1和pGenesil-1-siAFP2。酶切鉴定和测序分析,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功。重组质粒有效转染HepG2细胞并显著抑制AFP表达。结论:成功构建具有活性的、能够表达shRNA靶向干扰AFP蛋白的2个重组质粒载体。Objective：To construct the recombinant plasmid expressing two AFP - targeted short hairpin RNA （shRNA） with pGenesil- 1 plasmids vector, and Detect the Activity of Recombinant Plasmid in HepG2 cells. Methods： Two pairs of DNA sequences were designed and synthesized, and formed complementary chains by annealing respectively. Then the obtained products, siAFP1 and siAFP2, were inserted into plasmid vector pGenesil- 1 with U6 promoter. The recombinant plasmids were transforming into the Escherichia coli strain DH5α for screening and amplifying. The sequence analysis of the plasmids and identified by SalI digestion was carried out. The biological activity of recombinant plasmid was detected by transfecting into the HepG2 cell line. Results ： The two AFP - targeted shRNAs expressing frame were successfully inserted into the pGensil - 1 plasmid vector respectively, and the obtained shRNAs coding sequences were consistent with the designed fragments. The recombinant plasmid inhibited effectively the expression of AFP gene in HepG2 cells. Conclusion ： The construc- tion of two recombinant plasmids of AFP - targeted shRNA was successful. Two recombinant plasmids expressing AFP - targeted shRNA inhibited effectively the expression of AFP gene in HepG2 cells.

关 键 词：AFP蛋白 RNAI SHRNA 重组质粒载体 

分 类 号：R392[医药卫生—免疫学]