Construction of a full-length iASPP expression plasmid pcDNA3.1(+)/iASPP and its biological activity

In order to obtain a full-length expression plasmid for the p53 inhibitor protein, iASPP, fractional amplification was used to clone its full-length coding sequence (CDS) region. The amplified PCR product was then digested and inserted into the pMD19-T simple vector and subcloned into the pCDNA3.1(+...

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Bibliographic Details
Published in:Plasmid 2009-07, Vol.62 (1), p.10-15
Main Authors: Liu, Ze-Jun, Gao, Xing, Cai, Yun, Yang, Xin, Fu, Xiao-Lan, Chen, Jie, Zhang, Xiao-Bing, Xin, Hai-Ming
Format: Article
Language:English
Subjects:
Apoptosis
Cell Line, Tumor
Cloning, Molecular - methods
Electrophoresis
Expression plasmid
Genetic Vectors - genetics
Humans
iASPP
Immunoprecipitation
Intracellular Signaling Peptides and Proteins - genetics
Intracellular Signaling Peptides and Proteins - metabolism
Open Reading Frames - genetics
p53
Plasmids - genetics
Polymerase Chain Reaction
Recombinant Proteins - metabolism
Repressor Proteins
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Summary:In order to obtain a full-length expression plasmid for the p53 inhibitor protein, iASPP, fractional amplification was used to clone its full-length coding sequence (CDS) region. The amplified PCR product was then digested and inserted into the pMD19-T simple vector and subcloned into the pCDNA3.1(+) vector. A recombinant eukaryotic expression vector containing the complete CDS region of iASPP was successfully constructed. pcDNA3.1(+)/iASPP was able to express iASPP protein in an in vitro translation system and in cells. Its biological activity was verified using Western blotting, immunoprecipitation and cell apoptosis analysis. This successful preparation of a full-length iASPP expression plasmid lays the foundations for further studies on the function of iASPP.
ISSN:0147-619X
1095-9890
DOI:10.1016/j.plasmid.2009.01.004