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(+) 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.
Liu
Ze-Jun
ZJ
Southwest Cancer Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, People's Republic of China. zejunliu2000@yahoo.com.cn
Gao
Xing
X
Cai
Yun
Y
Yang
Xin
X
Fu
Xiao-Lan
XL
Chen
Jie
J
Zhang
Xiao-Bing
XB
Xin
Hai-Ming
HM
D017209:Apoptosis; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D004586:Electrophoresis; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins
D047908:Intracellular Signaling Peptides and Proteins; C119612:PPP1R13L protein, human; D011994:Recombinant Proteins; D012097:Repressor Proteins