Construction of eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein
- VernacularTitle:PIAS3的Myc融合蛋白真核表达重组质粒的构建
- Author:
Jiang LI
;
Yuanli ZHEN
;
Nanyang YANG
;
Junfang ZHANG
;
Zhongnan WANG
;
Xiaomeng LI
- Publication Type:Journal Article
- Keywords:
PIAS3;
Myc fusion protein;
recombinant plasmid
- From:
Journal of Jilin University(Medicine Edition)
2006;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein and express the fusion protein Myc-PIAS3.Methods The full length PIAS3 fragment of 1 851bp was amplified by PCR and ligated into pMD18-T vector. The full length PIAS3 fragment was subcloned into eukaryotic pCMV-Myc vector between SalⅠ and NotⅠ sites.The recombinant pCMV-Myc-PIAS3 plasmid was trandfected into PC3 cells.The eukaryotic expression Myc-PIAS3 protein was checked by Western blotting with Myc antibody.Results The recombinant plasmid showed right sequence by the full length sequencing.The recombinant plasmid of pCMV-Myc-PIAS3 was identified by enzyme digestion.As expected,by EcoRⅠ digestion,it showed two bands of 4 357bp and 1 318 bp. By XbaⅠdigestion,it showed two bands of 3 291 bp and 2 384 bp.The sequencing result showed a N-terminal Myc of 13 amino acids followed with PIAS3 gene sequence in right reading frame.The pCMV-Myc-PIAS3 plasmid was transfected into prostate cancer PC3 cells.A specific protein expression band at relative molecular mass 68 000 was obtained by using Myc-antibody with Western blotting method.Conclusion The recombinant plasmid of pCMV-Myc-PIAS3 is sucssesefully constructed,and Myc-PIAS3 fusion protein is sucssesefully expressed.
