Cloning And Expression Of GILT And COX-2 Genes From Japanese Eel Anguilla Japonica

The Japanese eel(Anguilla japonica)is one of the world's major aquaculture species.It is an important economic fish in China.But the research on mechanism of the immune response is relatively scarce.In this paper,we cloned two genes with immune function from the Japanese eel,GILT and COX-2.GILT plays a key role in the processing and presentation of MHC class II-restricted antigen(Ag),thus unfolding native protein Ag and facilitating subsequent cleavage by proteases.COX-2 are involved in the biosynthetic pathway of prostaglandin and thromboxanes synthesis,as they catalyze the initial conversion of arachidonic acid to prostaglandin endoperoxides G2 and H2.In this study,we searched the expression profiles of GILT and COX-2 in glass eel and multiple tissues from na?ve and LPS?Poly I:C and Edwardsiella tarda challenged Anguilla japonica elver were investigated.In addition we use Luciferase assay to test the promoter activity of GILT.The results will enrich the base immunologic knowledge of Japanese eel and will be helpful for controlling and preventing the bacterial diseases of Japanese eel.(1)We clone two GILT genes homologue,designated as Aj GILT-1 and Aj GILT-2.The open reading frame(ORF)of Aj GILT-1 consists of 765 bases,encoding a protein of 255 amino acids.The Aj GILT-2 c DNA is 843 bp in length and includes an ORF of 780 bp encoding a putative 260-peptide.The deduced proteins possesses the typical structural feature of known GILT proteins,including an active-site motif CXXC,a GILT signature sequence CQHGX2ECX2NX4 C,and 11 conserved cysteines.Analysis of Aj GILT-1 and Aj GILT-2 genomic organization revealed seven exons interrupted by six introns,which were similar to that in other vertebrates.There are binding sites which are related to interferon in the promoter sequences of Aj GILT-1 and Aj GILT-2.By detecting the activity of promoter fragments of Aj GILT-2 shows that there may be an important transcriptional binding sites between-1006~-512.The result of real-time PCR showed that Aj GILT-1 m RNA was expressed in sexual gland,head kidney and spleen.While Aj GILT-2 was mainly expressed in mucosal immune system--gill,skin,intestine.Upon intraperitoneal injection with LPS and E.tarda,the expression of Aj GILT-2 was significantly boosted in skin.It reached the peak at 16 h injected with LPS in skin.Moreover,a significantly up-regulated expression of Aj GILT-2 in intestine was respectively found at 24 h challenged with Poly I:C.We also find that the transcript level of Aj GILT-2 in glass eel was significantly up-regulated at 24 h which treated with Edwardsiella tarda.These results suggested that GILT may play an important role in innate immunity of Japanese eel against bacterial infection.(2)A full-length c DNA for COX-2(Aj COX-2)was successfully cloned from the Japanese eel(Anguilla japonica)by PCR and RACE.The predicted protein of Aj COX-2 is 606 amino acids in length,which contained all the conservative structural domains and the active sites for both the peroxidase and cyclooxygenase as in its vertebrate homolog.In physiological condition,Aj COX-2m RNA was highly expressed in gill,skin and fish blubber in Japanese eel elver.Additionally,a significant increasing m RNA expression of Aj COX-2 was detected in both skin and fish blubber(P <0.05)at 8 h after intraperitoneal injection with LPS and poly I:C,whilst a significant up-regulated m RNA expression of Aj COX-2 was found only in fish blubber at 8 h after challenged by Edwardsiella tarda,a pathogenic bacterium isolated from Japanese eel.The results suggested that Aj COX-2 might be an early immediate gene in response to the immune stimuli and involved in the innate immune response against the pathogens infection in Japanese eel.