| Background:Ulcerative colitis (UC),one of non-specific chronic inflammatory conditions of the gastrointestinal tract with unknown complex etiology,is a chronic inflammatory bowel disorder characterized by diffuse mucosal inflammation of the colorectum with exacerbations and remissions. The morbidity of UC in China is increasing year by year. Until now misdiagnosis and mistreatment of UC often happen in clinical practice due to lack of a gold standard for the diagnosis and effective treatment.Therefore,it is considered as a stubborn diseases by WHO. In recent years our team screens out differential expression of gene and protein by a sequence of previous study such as gene microarray, two-dimensional electrophoresis, mass spectrum analysis and so on.Our preliminary research revealed that the gene and protein of human heat shock factor2(HSF2) were increased expression in UC patients compared with the healthy comparers.Furthermore, immunohistochemical staining with HSF2in mucosal of UC patients is passtive correlation with severity of illness.Those results prompted that there was some correlation between HSF2and pathogenesis of UC.Therefore,the molecular biology research of HSF2will help to discover the roles of HSF2in the pathogenesis of UC.Aim:To construct a eukaryotic expression plasmid encoding the HSF2gene and to examine its expression and localization in Caco-2cells, a human colon adenocarcinoma cell line using FLAG tag as a reporter.METHODS:The coding sequence of the HSF2gene was amplified of HSF2Human cDNA ORF clone by PCR and subcloned into pCMV-Myc vector after digestion with EcoR I and Knp I. After the identity of recombinant plasmid was verified by direct sequencing, the plasmid was transfected into Caco-2cells by Lipofectamine means. Total cellular RNA was extracted and reverse transcribed into cDNA,then tested by PCR.The expression of the HSF2and recombinant fusion protein in Caco-2cells were detected by Western blot. The expression and localization of the HSF2and recombinant fusion protein in Caco-2cells were observed by laser scanning confocal microscopy.Results:The coding sequence of the HSF2gene was inserted into the pCMV-Myc vector successfully. Restriction enzymes digestion showed that the length of the insert was1557bp, matching the expected size. The mRNA lever of HSF2in recombinant plasmid transfected cells was higher compared with control and blank vector ones.The expression of HSF2-FLAG recombinant fusion protein, which had a molecular weight of70kDa, was detected by Western blot. Meanwhile, The expression of HSF2in recombinant plasmid transfected cells increased dramaticlly in contrast with matched groups.The HSF2and HSF2-FLAG recombinant protein were localized predominantly to the cytoplasm but partially aggregated around the nuclear envelope of Caco-2cells.Conclusion:Recombinant plasmid pCMV-HSF2-FLAG has been successfully constructed, and this is the basis for further studies of the possible roles and mechanisms of HSF2in ulcerative colitis. |
