摘要
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,PstⅠ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TKshRNA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。
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-shRNA1,hMGFP-shRNA2,hMGFP-shRNA3,hMGFP-shRNA4） were designed.The four recombinant plasmid were identified by PstⅠrestriction enzyme.Then the four plasmids were transfected into mouse hepatoma cell line Hepa1-6 for shRNA screening by reverse transcription-polymerase chain reaction and TK enzyme activity analysis.Results The four shRNA recombinant plasmids were correct verifying by PstⅠrestriction enzyme.shRNA screening identified that hMGFP-shRNA1 exerted the most significant inhibition effect on TK gene.Conclusion The recombinant plasmid of TK-targeted shRNAs is successfully screened.
出处
《新乡医学院学报》
CAS
2011年第4期406-409,共4页
Journal of Xinxiang Medical University
基金
国家自然科学基金面上项目(编号:30870871)
