大鼠Kir6.2基因真核表达载体的构建及其在HEK293细胞中的表达

Construction of a eukaryotic expression vector containing the Kir6.2 gene and its expression in HEK293 cells

目的克隆Kir6.2基因,构建真核表达载体pcDNA3.0/Kir6.2,将重组质粒转染HEK293细胞获得高表达Kir6.2基因的细胞克隆。方法从SD大鼠脑组织中提取总RNA,采用RT-PCR方法获得Kir6.2基因的全长cDNA,插入T1-simple克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.0,利用脂质体将重组质粒转染HEK293细胞,经G418筛选获得抗性细胞克隆,采用RT-PCR和Western blot方法,鉴定Kir6.2基因在HEK293细胞中的过表达。结果经PCR和DNA序列测定证实,目的基因已插入重组质粒,RT-PCR和West-ern blot证明,经G418筛选得到的转基因HEK293细胞克隆中存在Kir6.2基因的表达。结论成功构建Kir6.2基因的真核表达载体,获得稳定表达Kir6.2基因的HEK293细胞克隆。

Objective：To clone the Kir6.2 gene,construct its eukaryotic expression vector,and obtain positive HEK293 cell clones stably expressing the Kir6.2 gene.Methods：Total RNA was extracted from the SD rat brain.Full-length cDNA encoding the Kir6.2 gene was obtained by RT-PCR and inserted into the T1-simple cloning vector.After the sequencing was confirmed,the gene was subcloned into pcDNA3 to construct the recombinant eukaryotic expression vector pcDNA3.0/Kir6.2.The recombinant plasmid was transfected into HEK293 cells by lipofectamin and positive cell clones were screened with G418.Expression of the Kir6.2 gene in the transfected cells was confirmed by RT-PCR and Western blot.Results：PCR and sequencing showed that the target gene was cloned into the recombinant vector.Overexpression of the Kir6.2 gene in the transfected HEK293 cells was identified by RT-PCR and Western blot.Conclusion：The eukaryotic expression plasmid containing the Kir6.2 gene was successfully constructed.Positive HEK293 cell clones stably expressing the Kir6.2 gene were obtained.