Cloning And Expression Analysis Of The Nf-κB Inhibitor IκBα Of Ayu (Plecoglossus Altivelis)

The full-length cDNAs of the Plecoglossus altivelis alpha inhibitor of NF-κB(PaIκBα) wasobtained using RACE（Rapid Amplification of cDNA Ends）. The full-length cDNA of PaIκBα is1341bp containing a5’untranslated region (UTR) of64bp,3’untranslated region (UTR) of341bpand open reading frame (ORF) of936bp encoding a polypeptide of311amino acids. The deducedanimo acids sequence of PaIκBα shared95%homology with Osmerus mordax, and shared76%,75%,70%,68%homology with Salmo salar, Oncorhynchus mykiss, Nile tilapia, Siniperca chuatsirespectively through homology. PaIκBα was found to have great homology with other IκBαs viamultiple alignments by vector NTI Suite7.0, containing a conserved degradation unit for DSGLESwith two hydrophilic amino acids L and E instead of serine35and serine39, which was thephosphorylation site; there were five ankyrin repeat domain containing length of about30aminoacids in the middle portion of PaIκBα; a PEST sequence in the C-terminus, including P (proline), E(glutamic acid), D (aspartic acid) and S(serine acid). The basic characteristic analysis usingProtParam software on PaIκBα protein showed that the oretical isoelectric point (pI) of PaIκBαwas4.83and calculated molecular weight was35137.2. On the amino acid composition, thenumber of leucine was maximum, followed by glutamate and other amino acids were less. Totalnegatively charge of amino acid residues and positive charge of the amino acid residues were50and25, respectively. The instability index of PaIκBα was49.82, belonged to the unstable protein.The hydrophilicity analysis of PaIκBα was87.49, which was classified as hydrophilic protein. TheTMHMM program analysis on PaIκBα showed that it didn’t exist transmembrane structure. TheSOPMA analysis on PaIκBα secondary structure showed that it was composed of α-helix, randomcoil, β-sheet and β-turn. EpiLoc subcellular localization analysis showed that PaIκBα protein waslocated in the cytoplasm. The phosphorylation sites prediction of PaIκBα by ExPASy NetPhosshowed that PaIκBα had eleven Ser sites, one Thr locus and five Tyr sites. The Three-Dimensionalstructure prediction of PaIκBα by SWISS-MODEL showed the spatial α-helix structure ofsweetfish PaIκBα was arranged parallelly through the β-sheet in the space, with the α-helixstructure having characterized structural sequence-five ankyrin repeats. Phylogenetic analysisrevealed that the IκBα of sweet fish which having obvious ethnic specificity and Osmerus mordax, Salmo salar, Oncorhynchus mykiss, Nile tilapia, Siniperca chuatsi were in the same phylogeneticbrench according to the species relationship between these five main branch. The degree ofevolution of IκBαs were consistent with species classification status, suggesting that nucleartranscription inhibitory factor from different species were conservative in the evolution. Theanalysis of PaIκBα distribution in different organizations of ayu by One-step RT-PCR showed thatthe expression level of PaIκBα in liver, kidney, spleen and gills were higher, followed in intestine,brain and muscle and least in heart. After infection of hydrophila aeromonas, sobria bacteria,Vibrio anguillarum and Vibrio parahaemolyticus, PaIκBα in ayu liver presented the trend oftime-dependent. The amount of PaIκBα expression increased gradually, reaching a maximum andthen declined in the post-transcriptional level. PaIκBα was very sensitive to lipopolysaccharide,with the expression achieving the maximum in2h after LPS stimulation than the control group.The impact of sodium chloride on PaIκBα is not obvious because the PaIκBα expression in testgroup increased significantly than the the control group only in24h with no significant differencein other time point. The transcriptional level of PaIκBα in test group rose after the ayu were fedwith astragalus polysaccharide for three days and reached the maximum value on the ninth day.The transcriptional level of PaIκBα in test group increased significantly compared to the controlgroup, and then the transcription of PaIκBα was lowered gradually. The expression patterns ofPaIκBα by semi-quantitative detection in ayu which were infected by bacterial, lipopolysaccharide,sodium chloride stimulation and fed with APS indicated that PaIκBα may play an important role inayu immune process.