Establishment And Application Of NF-κB-responsive D2EGFP Reporter System

NF-icB(nuclear factor kappa B) is an important transactivator, whose overactivation is a key point to cause and develop many kinds of diseases. So, it is one of very important and prospective study to detect and screen antagonistic drugs of NF-KB as a target.How to report and detect NF-KB activation and it's activated degree reliably and sensitively is the prerequisite for inhibiting NF-kB overactivation accurately , evaluating it's antagonistic drugs and researching signal transduction pathway related to NF-icB .Usually reporter gene system is applied to report the interaction between transcription factor and it's cis-element by detecting the reporter gene expression, such as Cat, Luc, GUS, LacZ, SEAP, etc. However, those enzyme reporter gene systems have a common defect that the reporter molecules are detected by certain substrate and cofactor to react, so the course is complicated and the results are not easy to be repeated and even the reporter molecules cannot be observed dynamically in cells. Consequently, screening drugs is fatal due to the defects.Green fluorescent protein (GFP) gene which is unique, new protein reporter gene analysed in vivo has more advantageous characters than enzyme reporter gene analysed in vitro, such as direct observation in situ, real time and whole kinetics, simple analysis , and so on. But wild type GFP and EGFP are not ideal to detect the transient changes of gene expression regulation. Hopefully, the new product-d2EGFP (destabilized EGFP) is more sensitive to detect the changes of gene transcription regulation than EGFP due to the factthat d2EGFP has a short fluorescence half-life of 2h, and low accumalation in cells . This characters give important hints on d2EGFP is an ideal reporter gene to study and detect NF-KB activation accurately. However, this study is in the early stage. The vector containing neor gene and SV40 minimal promoter and the stable transfected clonal cell lines have been not reported until now.In order to establish an ideal NF-KB activation-responsive d2EGFP reporter system, this experiment compared the-.enhancer ability of the man-made KB motif with that of the wild one, the fluorescent stability and sensibility of d2EGFP with that of EGFP , cultured clonal cell lines, and applied in screening drugs.The main results and conclusions were as follows:1. Three types of NF-KB-responsive GFP reporter gene vectors were constructed successfully with NF-KB cis-element KBx4 as enhancer , SV40 as mininal promoter or HIV with KBx2 motif as promoter ,d2EGFP and EGFP as reporter gene ,neor gene as selective gene.2. The time and dose effects of d2EGFP and EGFP induced by p65 protein were analysed after p65 vector transient contransfected with the three vectors respectively. It was demonstrated that p4KB-d2EGFP was the best NF-KB-responsive GFP reporter gene system among the three vectors.3. Stablely transfected p4KB-d2EGFP into HEK 293 cells and harvested positive clonal cell lines by G418 selection . The NF-KB-responsive d2EGFP clonal cell line named HEK-d2EGFP was established successfully.4. With the contransfection of NF-KB TFD(transcription factor decoy) and p65 vector into HEK-d2EGFP cells , the results showed the groups of 1 mg/L and 2mg/L TFD could antagonized the d2EGFP expression induced by p65 protein significantly. It was demonstrated that NF-KB-responsive d2EGFP reporter system had the specificity and actual effect.5. It was suggested that the NF-KB-responsive d2EGFP reporter systemscould report and detect NF-KB activation and screen it's antagonistic drugs in certain degree, and might lay the model foundations for studying relevant transcription factors' activation.