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

ABSTRACT

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.