先天性长QT综合征KCNQ1基因定点突变的研究  

PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro

作　　者：李伟[1] 杨钧国[1] 任法鑫[1] 康彩练[1] 张守焰[1] 徐涛[2] 

机构地区：[1]华中科技大学同济医学院心血管病研究所 [2]华中科技大学生命科学与技术学院生物物理与生物化学研究所

出　　处：《中华心律失常学杂志》2005年第3期230-233,共4页Chinese Journal of Cardiac Arrhythmias

基　　金：国家自然科学基金资助项目(30170377);国家科技攻关计划项目(2002BA711A07)

摘　　要：目的利用聚合酶链反应(PCR)技术对长QT综合征(LQTS)KCNQ1基因进行定点突变的研究。方法利用PCR定点突变技术,首先设计两对引物(包含预定的突变),通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2EGFPKCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2EGFPKCNQ1基础上获得了KCNQ1cDNAC682T的突变体,测序结果表明在序列中发生了预期的突变。结论KCNQ1定点突变体的构建为进一步的功能研究奠定了基础。Objective To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. Methods The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. Two sets of primers were designed according to the sequence of KCNQ1 cDNA, and mismatch was introduced into primers. Mutagenesis was performed in a two-step pCR. The amplified fragments from the third pCR which contained the mutation site were subcloned into the T-vector pCR2.1.Then the fragments containing the mutation site was obtained from pCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES_2-EGFP-KCNQ1. With Effectene transfection reagent, pIRES_2-EGFP-KCNQ1 was transfected into HEK cell. Results The sequencing analysis showed that the mutation site was correct. Mutation from T to C in 682 site of KCNQ1 cDNA was found. Conclusion These results may shed light on further functional study of KCNQ1 gene.

关 键 词：先天性长QT综合征 基因定点突变 长QT综合征(LQTS) 聚合酶链反应(PCR) HEK293细胞 KCNQ1基因 定点突变技术 真核表达载体 PCR扩增 突变位点 转染试剂 cDNA 功能研究 突变体 T载体 突变点 片段 

分 类 号：R541.1[医药卫生—心血管疾病] R971.1[医药卫生—内科学]