Screening of promoter DNA--binding protein of iNOS gene by phage display technique from human liver cDNA library

The sequence of iNOS promoter was identified in GenBank by bioinformatics based on the open reading frame (ORF) of iNOS gene and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was subcloned into pCAT3-Basic reporter vector, named as pCAT3-iNOSp. The HepG2 cell line was then transfected with pCAT3-Basic to serve as negative control, and pCAT3-promoter which contains the promoter region of CMV served as the positive control subject, and pCAT3-iNOSp served as the test subject, respectively. The choloraphenical acetyltransferase (CAT) activity was determined by enzyme linked immunosorbent assay (ELISA) kit. The T7 Select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque was performed to amplify and PCR products were sequenced. The expression of CAT in transfection of pCAT3-PS1TP1p was 4.2 times as higher as pCAT3-Basic plasmid. Sequence analysis was performed in 12 positive plaque, which were the iNOSp binding protein. The iNOS gene promoter identified in this study has shown to have transcription activity, and iNOS promoter DNA-binding proteins have been screened. The results will be useful for further study of the expression and regulation mechanism of iNOS in liver cell.