作　　者： ; ; ; ; ; ; ;

机构地区： 泸州医学院

出　　处： 《泸州医学院学报》 2009年第3期209-213,共5页

摘　　要： 目的:KChIPs作为瞬时外向钾通道(Ito)最重要的辅助亚基,在调控通道基因表达和电流特性中起重要作用。本实验拟构建人心房肌KChIP2基因表达质粒,为研究KChIP2对Ito的调控功能奠定基础。方法:①提取人心房肌总RNA,逆转录得到cDNA;②通过特异性引物进行PCR扩增得到KChIP2编码区全长序列,亚克隆到pMD18-T载体中,得到重组克隆质粒pMD18-T-KChIP2;③再设计带有酶切位点Hind Ⅲ和BamH Ⅰ的特异性引物,对重组克隆质粒pMD18-T-KChIP2进行PCR扩增,得到含有酶切位点的KChIP2编码区全长序列;④对含有酶切位点的KChIP2全长片段和pEGFP-c1进行Hind Ⅲ和BamH Ⅰ双酶切反应,然后进行电泳后胶回收,再进行连接和转化反应;⑤提取质粒,测序鉴定。结果:质粒测序结果与PubMed数据库KChIP2(AY026328)序列比对,同源性为99%。结论:成功构建人心房肌KChIP2基因表达质粒,为下一步转染实验研究KChIP2对瞬时外向钾通道的功能调控奠定了基础。 Objective： KChIP2 is the most important auxiliary subunits of transient outside potassium channel, which plays an important role in regulating the channel gene expression and the current characteristics. This study is to construct the expression plasmid pEGFP-KChIP2 of the human atrial myoeytes KChIP2 gene and establish the basis for researching the KChIP2 regulation function. Methods： ①The total RNA was extracted from the right atrial appendage and the cDNA was obtained through reverse transcription; ②The full length of the KChIP2 CDS region was obtained by PCR amplification with specific primers and the reconstructed cloning pinsmid pMD18-T-KChIP2 through subcloning with pMD18-T plasmid;③The specific primers including the enzyme cutting sites Hind Ⅲ and BamH Ⅰ were designed; acquire the the full length of the KChIP2 CDS region in cluding the enzyme cutting sites was constructed; ④The double enzyme cutting reaction containing the full length of the KChIP2 including the enzyme cutting sites and the pEGFP-cl plasmid was constructed; the electrophoresis products were reclaimed and the ligated reaction progressed; ⑤The ligated products were transferred to the competence cell DHSct and were cultured overnight; the reconstructed plasmid was extracted for sequencing and identificating. Results： Compared the sequence of the reconstructed plasmid with the data from the PubMed database （AY026328）, 661bp was the same as the database and the homology was 99%. Conclusion： The reconstructed expression plasmid was constructed successfully, which established the basis for further research on the regulation of KChIP2 for Ito.

关 键 词： 瞬时外向钾通道 心肌细胞 钾通道相关蛋白 基因克隆

领　　域： [生物学] [生物学]