The Gene Cloning And Expression Analysis Of Immune Gene CXCR4, IL-1β And Its Receptor(IL-R2) In Asian Swamp Eel Monopterus Albus

The cDNA of CXCR4a, CXCR4b, IL-1β and IL-1receptor2(IL-R2) genes of Monopterus albus were cloned using the method of rapid amplification of cDNA ends (RACE) in this study. Expression pattern of IL-1(3and IL-R2mRNA in different tissues and the inducection changes after injection with bacteria were also detected. The results were following:(1) The full-length cDNA of CXCR4a was isolated and it contains1312bp (GenBank accession no. JX122807). The CXCR4a cDNA contains1140bp of open reading frame (ORF), the deduced amino acid sequence contains379amino acid residues including three N-Glycosylation sites. The basic second structure of the protein can divide into three parts,41.69%α-Helix,11.6%β-sheet and46.8%0(others, include coil and turn). Protein3D structure prediction of CXCR4a showed that it has seven a-Helix consisting of a cylindrical structure. The analysis of amino acid sequence homology and multiple sequence alignment suggested that vertebrate CXCR4a proteins have four extracellular domain and seven transmembrane structure, with one cysteine at1st,2nd,3rd,4th extracellular domain, which may form disulfide bonds. CXCR4a has two cysteines in5th,7th transmembrane in almost all vertebrate. Three cysteines were found in the6th transmembrane in teleost while only one cysteine was found in the tetrapod. The phylogenetic tree showed that maCXCR4a clustered together with other fish CXCR4a. Homologous comparison showed that maCXCR4a and stickleback had the highest similarity, value of87.7%. Two extrons and one intron was found in the genome of maCXCR4a and the length of the intron is only319bp. In addition, the partial sequence of CXCR4b was obtained, with length of562bp.(2) maIL-1β cDNA full-length is879bp with open reading frame of741bp and encoding246amino acid residues (aa). IL-R2cDNA is1061bp with open reading frame of630bp and encoding209aa. The sequence of IL-1β in the DNA is2522bp with five exons and four introns. The similarity of maIL-1β protein and IL-1β in the sea bass reached at77%. Phylogenetic tree showed that maIL-1β belong to typeⅡ IL-1β. The similarity of maIL-R2protein and IL-R2in the Oryzias latipes was60.3%. Tissue expression showed maIL-1β was predominatly expressed in the liver and the fold relative to β-actin was0.63×10-2, followed by a higher expression in the brain, intestines and skin. IL-R2mainly expressed in the liver, skin and intestines. The expression of IL-1β and IL-R2were low in muscle, blood and heart. After injecting with Aeromonas hydrophila, the expression of maIL-1β significant increase in head kidney in1h and6h, with92-fold (p<0.05) and95-fold (p<0.01) respectively. The expression levels of IL-1β was enhanced to479-fold (p<0.01) at6h and87-fold at24 h (p<0.05). The bacteria can up-regulate the maIL-R2expression in head kidney and spleen at1h and6h after the injection (p<0.05), which expression fold change reached at910-fold at6h in head kidney (p<0.01). In vitro, head kidney cells of Asian swamp eel were stimulated with Aeromonas hydrophila and the expression of maIL-1β and maIL-R2was tested with real-time PCR. The results suggested maIL-1β expression only increased significantly atlh (p<0.01,21.7-fold) while IL-R2expression levels were significantly increased at1h (p<0.01,41-fold),6h and48h (p <0.01,42-fold). These result that show IL-1β and IL-R2were taken part in the immune response after infection with bacteria in the Asian swamp eel.