干扰AFP蛋白表达shRNA表达载体构建及活性鉴定.docVIP

干扰AFP蛋白表达shRNA表达载体构建及活性鉴定 　 作者：张国英 刘明社 赵中夫* 张芸 杨慧 武延隽 【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。 【关键词】 AFP蛋白　RNAi　shRNA　重组质粒载体 Abstract 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 construction 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. Key Words AFP；RNA interference；Short hairpin RNA；HepG2 cells 长期以来，人们只是把AFP作为一个肿瘤标志物，用于原发性肝癌(HCC)的诊断指标之一。诸多研究关注于检测甲胎蛋白（alpha-fetoprotein,AFP）对原发性肝癌诊断运用，而对AFP和肿瘤细胞发生及增殖的关系并不完全清楚［１～3］。近年发展起来的RNA干扰（RNA interference, RNAi）技术为探索AFP的生物学功能提供