Construction of recombinant plasmid pCMV-Myc-PIASx? and its protein expression / 吉林大学学报(医学版)

Objective To construct the recombinant plasmid pCMV-Myc-PIASX? and express the fusion protein in mammalian cells.Methods PIASx? fragment was digested from the original vector pGADT7 with SalⅠand NotⅠ,and then was inserted into the targeted pCMV-Myc vector by the recombinant DNA technique.After identification,the recombinant plasmid was transfected into CHO cells.The expression of recombinant Myc-PIASx? fusion protein was detected by Western blotting.Results By the restriction enzyme digestion,fragment purification,ligation and transformation,the recombinant plasmid was obtained.The right recombinant plasmid pCMV-Myc-PIAS3 was identified with enzyme digestion and sequencing.By EcoRⅠ digestion analysis,pCMV-Myc-PIASx? showed a 5641 bp band.By XbaⅠdigestion analysis,pCMV-Myc-PIASx? showed two expected band of 3291 bp and 2349 bp.A specific protein expression band at 68 000(PIASx? fusion protein) was showed in Western blotting,which matched recombinant plasmid.Conclusion The recombinant plasmid of pCMV-Myc-PIASx? is sucssesefully constructed,which provids a good tool for further function study on PIAS family.

Construction of eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein / 吉林大学学报(医学版)

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.