串联表达大鼠心肌细胞Kir2·1shRNA载体的构建及其体外效应  

Construction of vector tandem expressing rat ventricular myocyte Kir2.1 shRNA and its effect in vitro

作　　者：雷印胜[1] 张海洲[1] 王来城[2] 郭兰敏[1] 范全心[1] 邹承伟[1] 李红昕[1] 王安彪[1] 

机构地区：[1]山东大学山东省立医院心外科 [2]山东大学山东省立医院中心实验室,济南250021

出　　处：《中华医学杂志》2005年第41期2910-2915,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30471711)

摘　　要：目的构建串联表达5个针对大鼠kir2·1基因shRNA的真核表达载体,观察其对该基因表达的影响。方法选择5个针对大鼠kir2·1基因的RNA干扰位点,分别设计合成相应的5对寡核苷酸链,形成双链后分别连入带有U6启动子的相应载体,反复酶切连接将表达5个kir2·1shRNA的目的基因依次连接入载体pEGFP6-1,构建成串联真核表达载体pEGFP6-1Kir2·1,将其转染培养的大鼠心肌细胞,RT-PCR和Western印迹检测其对Kir2·1mRNA转录和蛋白表达的影响。结果成功构建串联表达5个大鼠Kir2·1基因shRNA的真核表达载体,该载体对大鼠心肌细胞Kir2·1mRNA转录的抑制率为83·5%,对心肌细胞Kir2·1蛋白表达的抑制率为68·1%。结论串联表达多个大鼠心肌细胞Kir2·1基因shRNA真核表达载体介导的RNA干扰可以特异性抑制该基因的表达,可能成为制造生物起搏器的一种新方法。Objective To construct a recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem and its effect in vitro Methods Ventricular myocytes were collected from newborn Wistar rats and cultured. Five sites targeting on the rat Kir2.1 gene were selected. Accordingly 5 pairs of oligonucleotide fragments were designed, synthesized, and annealed to obtain doublestranded DNAs. The 5 pairs of oligonucleotide were then cloned into the vector pGenesil-1 by repeated excision and ligation successively. The tandem recombinant vector pEGFP6-1Kir2. 1 was thus constructed and transfected into the cultured rat myocytes. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Kir2.1 in the myocytes. Sequence not related to Kir2.1 sequence with mismatched bases was designed and used as control. Results A recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem was constructed. 96 hours after the transfection RT-PCR showed that the Kir2.1 mRNA transcription was suppressed by 83, 6% , and Western blotting showed that the Kir2.1 protein transcription was suppressed by 68.1% in comparison with the control. Conclusion The vector that expresses the 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem is able to suppress the expression of Kir2.1 in rat ventricular myocytes. Application of such vector may be a new method to produce a new type of heart biological pacemaker.

关 键 词：基因疗法 kir2.1基因 串联表达 RNA干扰 

分 类 号：R346[医药卫生—基础医学]