干扰转酮醇酶的短发卡RNA表达载体的构建与筛选

摘要

Objective To screen the recombinant plasmid expressing TK-targeted short hairpin RNA( shRNA). Methods Aimed at TK gene order,four pairs of DNA sequences(hMGFP-shRNAl, hMGFP-shRNA2, hMGFP-shRNA3, hMGFP-shRNA4) were designed. The four recombinant plasmid were identified by Pst I restriction enzyme. Then the four plasmids were transfected into mouse hepatoma cell line Hepal-6 for shRNA screening by reverse transcription-polymerase chain reaction and TK enzyme activity analysis. Results The four shRNA recombinant plasmids were correct verifying by Pst I restriction enzyme. shRNA screening identified that hMGFP-shRNAl exerted the most significant inhibition effect on TK gene. Conclusion The recombinant plasmid of TK-targeted shRNAs is successfully screened.%目的 筛选出干扰小鼠转酮醇酶(TK)的短发卡RNA(shRNA)表达载体.方法 针对TK基因序列设计4条TK干扰序列:hMGFP-shRNA1、hMGFP-shRNA2、hMGFP-shRNA3、hMGFP-shRNA4,Pst Ⅰ酶切鉴定其序列.然后利用质粒转染小鼠肝癌细胞株Hepa1-6,用逆转录-聚合酶链式反应和酶活性方法检测转染TK shRNA后Hepa1-6细胞内TK mRNA和蛋白表达变化.结果 经酶切凝胶电泳证实shRNA载体构建正确,质粒筛选实验表明hMGFP-shRNA1质粒对TK基因的抑制作用最强.结论 成功构建表达靶向TK的shRNA重组质粒载体.