摘要
目的:对中国人遗传性长QT综合征2型(LQT2)HERG基因突变E505D进行体外定点突变研究,并构建有基因突变E505D的pCMV-script/HERG的真核表达载体。方法:采用PCR定点突变技术(重叠延伸法),根据突变位置附近的两个单一限制性内切酶位点(BamHI和EcoRI)设计两对引物,将突变位点设计在引物上,通过PCR扩增,使扩增片段上含有所需要的突变位点,最后将扩增片段克隆入pCMV-script载体中。结果:DNA测序结果表明,HERG基因在第1515位由G变成T,发生第505位谷氨酸→天冬氨酸的改变(E505D),定点突变成功。结论:PCR技术介导的定点突变准确、高效。pCMV-script/HERG(E505D)的成功构建,为进一步进行该基因突变的功能研究奠定了基础。
Objective： To perform site-directed mutagenesis of a new HERG mutation（E505D） identified in a Chinese family with Long QT syndrome and construct the recombinant expression plasmid pCMV-script/HERG containing mutant cDNA. Methods： Two pairs of primers were designed according to the restricted sites BamH Ⅰ and EcoR Ⅰ of the HERG sequence with mutant sites introduced into primers. Mutagenesis was performed in PCR, and the fragments amplified by PCR containing the mutant site were subcloned into the pCMV-script vector. Results： Sequence analysis confirmed the mutant site, indicating the successful induction of the mutation E505D in HERG gene. Conclusion： PCR based site-directed mutagenesis is accurate and efficient. The constructed recombinant expression plasmid pCMV-script/HERG（ES05D） may provide a molecular basis for further functional investigation of HERG mutation.
出处
《交通医学》
2008年第6期642-643,646,共3页
Medical Journal of Communications
