Construction of Adenovirus Vector Carrying Human HSP75 Gene and Expressing in C17.2 Neural Stem Cells
10.3969/j.issn.1006-9771.2015.03.005
- VernacularTitle:人HSP75 基因重组腺病毒载体构建及在C17.2 神经干细胞的表达
- Author:
Yan WANG
;
Qingzhuang CHEN
;
Deqi JIANG
;
Mingxing LI
;
Siyuan JIA
;
Ning ZHU
;
Yong WANG
- Publication Type:Journal Article
- Keywords:
heat shock protein 75, recombinant adenovirus vector, C17.2 neural stem cells
- From:
Chinese Journal of Rehabilitation Theory and Practice
2015;21(3):264-268
- CountryChina
- Language:Chinese
-
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 pHBAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant adenovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detected by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplification 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. Conclusion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.
