Title: Construction and expression of recombinant adenovirus carrying human sarco(endo)plasmic reticulum Ca2+ ATPase2a.
Abstract: Objective:To construct recombinant adenovirus vector bearing the human sarco(endo)plasmic reticulum Ca2+ ATPase2a and amplify the adenovirus in 293 cells.Methods:Human SERCA2a cDNA(3700bp)was cloned into adenovirus shuttle plasmid pAdtrackCMV to generate a recombinant plasmid pAdtrackCMV-SERCA2a,then the linearized plasmid was transformed into E.coli BJ5183 cells which had been electroporated adenovirus backbone plasmid pAdEasy-1.The identified recombinant plasmid was digested with PacⅠand transfected into 293 cells to package the adenovirus,green fluorescence protein expression was detected under fluorescent microscope.Co-cultured recombinant adenovirus and SD rat cardiomyocytes,SERCA2a protein was overexpressed by western-blot.Results:Recombinant adenovirus with SERCA2a was constructed successfully,which were effectively infected to SD rat cardiac muscle cell.Conclusion:The recombinant plasmid SERCA2a was established rapidly and simply by homologous recombination.It may be a new tool for gene therapy using SERCA2a.
Publication Year: 2010
Publication Date: 2010-01-01
Language: en
Type: article
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