Abstract:

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.

Key words: Alzheimer disease； Plasmids； Genes， kir6.2； HEK293 cell