Pcdna3.1 ( + ) /hfasl Construction Of Eukaryotic Expression Vector And Its Application In The Treatment Of Thyroid Eye Disease In An Animal Model

OBJECTIVE: To construct the recombinant eukaryotic expression vector pcDNA3.1(+)/hFasL and investigate its transformatiom and transient expression in COS-7 cells.METHODS: The full-length cDNA of hFasL was obtained from the prokaryotic plasmid vector pBluescript II KS(+)/hFasL cutted with Xba I and was then subcloned into the multiple cloning site of the eukaryotic expression vector pcDNA3.1(+). The recombinant eukaryotic expression plasmid pcDNA3.1(+)/hFasL was identified with restriction enzyme digestion analysis and DNA sequencing. It was then transformed into the COS-7 cells by Lipofectin Reagent and its expression was detected by immunocytochemical staining.RESULTS: The full-length sequence of hFasL(0.97kb) obtained from pBluescript II KS(+)/hFasL was identical with what released in GeneBank. Restriction enzyme digestion analysis with XbaⅠ,DraⅡ,HindⅢand DNA sequencing confirmed the correct construction of the recombinant plasmid of pcDNA3.1(+)/hFasL. Immunocytochemical staining showed the expession of hFasL on COS-7 cells.