AK3Gene Analysis Of Overexpression In Cells

Objective The study aims to construct recombinant plasmids of pcDNA3.1-AK3-FLAGand investigate whether protein interact with Sedlin in mammalian cells and invitro by immunofluorescence,co-immunoprecipitation and GST pulldown assay.Methods The upstream and downstream primers of AK3were designed, thefull-length cDNA sequence of AK3was amplified by PCR,and consequently insertedinto eukaryotic expression vector of pcDNA3.1.The correct recombinant plasmids ofpcDNA3.1-AK3-FLAG,identified by specific restriction enzymes were sent toSANGON company for sequencing.In comparison with BLAST,the correct plasmidspcDNA3.1-AK3-FLAG was transfected into COS7cells to observe their spatialdistribution,then co-transfection into COS7cells to observe the co-localization withSedlin.Co-immunoprecipitation assay was performed to investigate the interactionbetween them with transfection of the plasmid pCDGFP-Sedlin andpcDNA3.1-AK3-FLAG. The plasmid pGEX-5X-3-Sedlin was transformed into BL21competent cells to prepare fusion protein, and transfected pCDGFP-Sedlin intoHEK-293T cells, GST pulldown assay was performed to confirm the direct interactionbetween AK3and Sedlin protein.Results The recombinant plasmids of pcDNA3.1-AK3-FLAG was successfullyconstructed by restriction enzyme digestion and sequencing.Sedlin protein wasrespectively distributed in cell nucleus and cytoplasm.and there existed overlappingrelation under the fluorescence microscope.Expression of AK3protein in HEK-293T cells was observed by Western blot assay,AK3-FLAG precipitated GFP-Sedlin proteindemonstrated by Co-immunoprecipitation assay.GST-Sedlin fusion protein wassuccessfully expressed in appropriate concentration of IPTG, AK3-FLAG protein couldnot pull down Sedlin protein verified by GST pulldown assay in vitro.Conclusion The result of indirect immunofluorescence experiment demonstrated thatthere was no colalization of AK3and Sedlin in mammalian cells.Co-immunoprecipitation and GST pulldown experiments indicated that AK3proteincan not interact with Sedlin in vivo and in vitro. Objective This study aims to construct recombinant plasmids ofpCDGFP-SEDL-D47Y,pCDGFP-SEDL--S73L and investigate whether protein interactwith EBI3protein in mammalian cells and in vitro.Transfected into COS7cells toobserve them single localization difference with the wild type, then co-transfectedwith EBI3protein with Flag tag into COS7cells together to observe in mammalsco-localization of cells.Methods The upstream and downstream primers of SEDL-D47Y and SEDL-S73Lwere designed, and consequently inserted into eukaryotic expression vector ofpCDGFP.The correct recombinant plasmids of pCDGFP-SEDL-D47Y,pCDGFP-SEDL--S73L and identified by specific restriction enzymes were sent toSANGON company for sequencing.In comparison with BLAST,the correct plasmidswere transfected into COS7cells to observe their spatial distribution,thenco-transfection into COS7cells with FLAG-EBI3to observe the co-localization.Results The recombinant plasmids of pCDGFP-SEDL-D47Y，pCDGFP-SEDL--S73L were successfully constructed by restriction enzyme digestion and sequencing.pCDGFP-SEDL-D47Y protein and EBI3protein were respectively distributed inperinuclear and nucleus.and there existed overlapping relation under the fluorescencemicroscope.pCDGFP-SEDL-S73L protein and EBI3protein were respectivelydistributed in perinuclear. Conclusion The result of indirect immunofluorescence experiment demonstrated thatSEDL-D47Y protein can co-locate with EBI3in nucleus and perinuclear and thatSEDL-S73L protein can co-locate with EBI3protein in perinuclear of the cells.