先天性长QT综合征2型hERG基因G604S突变真核表达载体的构建和表达  

Construction and expression of congenital LQT2 relating hERG gene G604S mutation eukaryotic expression vector

作　　者：韩稳琦[1] 霍建华[1] 李国良[1] 蒋永荣[1] 武金娥 孙超峰[1] 

机构地区：[1]西安交通大学第一附属医院心内科,西安710061

出　　处：《郑州大学学报（医学版）》2015年第6期753-757,共5页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30801133

摘　　要：目的:构建长QT综合征(LQTS)2型h ERG基因G604S突变的真核表达载体G604S-h ERG-pc DNA3、G604S-h ERG-p EGFP。方法:采用重叠延伸PCR法在p EGM-h ERG基础上构建G604S突变体PGEM-h ERG-G604S,通过限制性内切酶法和基因重组技术将突变体插入到真核表达载体pc DNA3和p EGFP-C2中并测序验证,用脂质体转染法将G604S-h ERG-p EGFP转染至HEK293细胞并观察其荧光表达。结果:构建的含有突变位点的真核表达载体经DNA测序均成功验证h ERG基因1 810位点碱基G突变为A,构建的G604S-h ERG-p EGFP在HEK293细胞中成功表达绿色荧光。结论:成功构建h ERG基因G604S-h ERG-pc DNA3和G604S-h ERG-p EGFP表达载体。Aim：To construct eukaryotic expression vectors G 604S-hERG-pcDNA3 and G604S-hERG-pEGFP linked to LQT2.Methods：PGEM-hERG-G604S containing G604S fragment was constructed using pEGM-hERG as a template by overlap extension PCR and then validated by DNA sequencing , then hERG-G604 S sequence was subcloned into pcDNA 3 and pEGFP-C2 vectors using restriction enzymes and gene recombination technology , respectively.After sequencing, G604 S-hERG-pcDNA3 and G604 S-hERG-pEGFP expression vectors were transfected into HEK 293 cells to obtain heterolo-gous expression system .Results：G604 S-hERG-pcDNA3 and G604 S-hERG-pEGFP eukaryotic expression vectors were con -structed successfully ,a missense mutation in hERG 1 810 site nucleotide G mutant to A was identified using DNA sequen -cing,hERG mutant was correctly combined to eukaryotic expression vector pEGFP -C2and expressing green fluorescent pro-tein confusion mutant G604S in HEK293 cells.Conclusion： The protocol can be used to construct the eukaryotic expres-sion vector G604S-hERG-pcDNA3 and green fluorescent protein expression vector G 604S-hERG-pEGFP.

关 键 词：先天性长QT综合征 HERG基因 突变 真核表达载体 

分 类 号：R541[医药卫生—心血管疾病]