重叠PCR法构建人心房肌SK2(KCNN2)基因表达质粒及鉴定和序列分析

Construction and identification of the expression plasmid of SK2 (KCNN2) gene from human atrial myocytes with Overlapping PCR

目的:小电导钙激活钾通道亚型2(SK2)在心房肌功能活动中起重要作用,但是由于其表达密度低,直接进行RT-PCR一步法无法得到该基因(KCNN2)的编码区全长序列,本研究旨在采用Overlapping PCR(重叠PCR)法进行基因全长序列的扩增和表达质粒的构建,探讨其在长片段基因扩增的应用。方法:收集人心房肌标本,采用提取总RNA之后逆转录为cDNA,分三段设计KCNN2基因(AY258141)引物进行分段扩增,同时进行分段测序,然后采用Overlapping PCR得到KCNN2基因编码区全长序列,通过限制性酶切位点定向克隆到表达载体pIRES-hrGFP上。采用酶切法和测序法进行鉴定。结果:三段KCNN2基因扩增产物大小与预测值一致,最后得到的表达质粒测序结果与基因库数据基本一致。结论:成功构建人心房肌SK通道基因表达质粒pIRES-hrGFP-SK2,Overlapping PCR能够很好的用于长片段基因扩增。

Objective： Small conductance calcium activated potassium channels type 2 （SK2） play a crucial role in atrial repolarization. It is difficult to acquire the full-length of its coded gene KCNN2 by RT-PCR with one step. We aim to get the full-length of KCNN2 gene and construct the plasmid by Overlapping PCR, and further more discuss the application of Overlapping PCR. Methods： Total RNA was extracted from human right atrial tissue and cDNA was acquired with reverse transcription, overlapping PCR was conducted with three pairs of primers which were designed according to the sequence of KCNN2 （AY258141） gene. The expression plasmid of pIRKS-hrGFP-SK2 was constructed by directed cloning with restriction enzyme site and identified by enzyme cutting and sequencing. Results： Three parts of PCR amplification were consistent with predicted size. The sequence of the plasmid was consistent with the gene-bank data except two sites, however, which were the same as gene in different tissues. Conclusion： The expression plasmid plRKS-hrGFP-SK2 was constructed successfully, overlapping PCR is a good choice for amplifying these genes with long size or low expression.