Construction of Adenovirus Vector Carrying Human HSP75 Gene and Expressing in C17.2 Neural Stem Cells / 中国康复理论与实践

ABSTRACT

Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pH-BAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant ad-enovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detect-ed by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplifica-tion revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×1010 PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclu-sion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.