1. Construction of a full-length iASPP expression plasmid pcDNA3.1+/iASPP and its biological activity
- Author
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Xin Yang, Xing Gao, Jie Chen, Yun Cai, Ze-Jun Liu, Xiao-Lan Fu, Xiao-Bing Zhang, and Hai-Ming Xin
- Subjects
Electrophoresis ,Expression vector ,Immunoprecipitation ,Genetic Vectors ,Intracellular Signaling Peptides and Proteins ,Repressor ,Apoptosis ,Biology ,Molecular biology ,Polymerase Chain Reaction ,Recombinant Proteins ,law.invention ,Blot ,Repressor Proteins ,Open reading frame ,Open Reading Frames ,Plasmid ,law ,Cell Line, Tumor ,Recombinant DNA ,Coding region ,Humans ,Cloning, Molecular ,Molecular Biology ,Plasmids - Abstract
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.
- Published
- 2009