Construction Of Eukaryotic Expression Vector Containing KIR6.2 Gene And Its Expression In HEK293 Cells

Objective:Potassium channels is a widespread membrane protein families and ATP sensitive type potassium channels (ATP-sensitive potassium channels KATP) widely distributed on different kinds of organization, plays an important role in a variety of physiological actions, KATP consists of two different types of protein subunits (Kir6. X and SURx) composed of eight polymer (4:4) complex. Different potassium channels subunits have a corresponding change in the brain of patients with Alzheimer's disease. But the specific mechanism of Kir6.2 in alzheimer's disease occurrence and development is not clear.This study aims to clone Kir6.2 gene, construct its eukaryotic expression vector and obtain positive HEK293 cell clones expressing Kir6.2 gene stably.Methods:1. The total RNA was extracted from SD rat brain. The full-length cDNA encoding Kir6.2 gene was obtained by RT-PCR method and preliminaryly appraisaled by 0.8% AGAR sugar gel electrophoresis. Recycled purpose PCR products by rubber cutting.2.Inserted the recycled PCR products into T1-simple cloning vector. take positive cloning for sequence determined 3. After the sequencing was confirmed, double digested pEASY-T1/Kir6.2 with restriction enzymes of Hindâ…¢and BamHâ… . Purify and recycle purpose extract about 1.2 Kb size. Plasmid pcDNA3.0 also double digestyed by restriction enzymes of Hindâ…¢and BamHâ… . Purify and recycle larger pieces cut. Use T4 DNA connection enzyme connect purpose extract and linearization empty plasmid pcDNA3.0. Use connected product transform DH5 alpha feeling modal bacterial. Clone Kir6.2 gene by colony PCR to identificate positive bacteria cloning. Electrophoresis analysis PCR product and extraction plasmid.4.The recombinant plasmid pcDNA3.0/Kir6.2 was transfected into HEK293 cells by lipofectamin method. Transfected with empty plasmid pcDNA3.0 and no transfected cells as contrl. G418 screen transfected cells get G418 resistance cells clones. Confirm overexpression of Kir6.2 gene in the transfected cells was with RT-PCR and Western blot.Results:1. RT-PCR product electrophoresised by 0.8% agarose gel received a peculiar stripe about 1.2 Kb.2. Contrast Sequencing result of recombinate cloning vector pEASY-T1/Kir6.2 and Kir6.2 sequence logged in Gene bank, Base sequences identically the same. Which proved the research was correctly cloned cDNA sequence of Kir6.2 gene.3. Confirmed by PCR and DNA sequencing, Kir6.2 purpose gene has been successfully inserted restructuring plasmid pcDNA3.0/Kir6.2.4. Extracted total RNA form HEK293 cell clones transfected with pcDNA3.0/Kir6.2. Received a peculiar stripe about 1.2 Kb by RT-PCR. While the control cell clones got no peculiar stripe after RT-PCR. HEK293 cell clones transfected with pcDNA3.0/Kir6.2 has the high expression Kir6.2 protein channel on 40kD place confirmed by Western Blot. The control groups only have extremely low levels of endogenous kir6.2 protein expression. Both indicate that the HEK293 cell clones carrying pcDNA3.0/Kir6.2 exist Kir6.2 gene.Conclusion:The eukaryotic expression plasmid containing Kir6.2 gene was successfully constructed. The positive HEK293 cell clones expressing Kir6.2 gene stably were obtained.Significance:Success construction of cell clones high express Kir6.2 gene may be a promising cell model for studying the biological function of the Kir6.2 gene and the role of Kir6.2 in the pathogenesis of Alzheimer disease.