Cloning, Bioinformatic Analysis And Differential Expression After Lps's Effection Of Rlj-ⅠκB

Nuclear factor NF-κB which is an impotrant transcripitional factor widely exists in the eukaryotic cells. It involves in cell proliferation and differention. However its excessive activation causes immuno-inflammatory response and is associated wite the occurrence,development,invasion of the tumor. Under normal circumstances NF-κB binding its specfic protein IκB exists in the form of trimer in the cytoplasm, this NF-κB is nonactiviated. When IκB is phosphorated by IκK and degenerated, NF-κB is activated into the nucleus to start the transcripiton of target genes.Therefore whether IκB is phosphorated or ot plays a vitol role in the regulation of NF-κB activity. At present the fuction and mechanism of NF-κB and IκB in mammals is deeply studied, it indicates that they participate in many signaling pathway. However the studies in the lower animals remains in the blank.Lampetra japonica is the most ancient vertebrate which is between vertebrates ang invertebrates.Therefore making research into the function of NF-κB and IκB in the Lampetra japonica is helpful to further findout their mechanism and molecular evolution that contributes to study signal transduction pathway in the Lampetra japonica.Two EST sequences of IκB are found in the cDNA library.Then I degined a primer to obtain CDS domain of IκB and its mRNA by 3'RACE and 5'RACE experiments named Lj-IκB. I designed degenerate primers based on conservative domain of NF-κB and obtained a NF-κB sequence. Both of them have a high homology by compared with with other species but it needs further cloning to get the gene CDS length.Bioinformatic analyzed the physical and chemical properties, structural characteristics of Lj-IκB. The results indicated that it has the IκB family specific ankyrin repeats. The Lj-IκB to thirteen species of animo acid sequence alignment showed that the whole species had a high homology. I also used real-time quantitative PCR probe to detect the Lj-IκB gene expression differences in the various tissues and organs of healthy Lampetra japonica. After LPS stimulation the expression levels in the heart, kidneys, intestines and muscle decresed while in the white blood cells, liver and gills the levels incresed. The results indicated that white blood cells, liver and gills are related to innate immunity.