Molecular Cloning, Characterization, Tissue Distribution Of Interferon And Nuclear Factor 45 Gene From Grass Carp (Ctenopharyngodon Idellus) And The Expression Analysis Of Interferon In Saccharomyces Cerevisiae

Here, we do some research about two importent immune-related gene :grass carp interfereon and NF45.In the part one, we report the cloning and characterization of the IFN gene from the grass carp (Ctenopharyngodon idellus). The 1191bp cDNA fragment harbouring the IFN gene exhibited one open erading frame (ORF) composed of 543bp flanked by a 34bp 5'-UTR and 614bp 3'-UTR. Grass carp IFN (gcIFN) is 181 amino acids in length, with the first 22 amino acids representing a putative signal peptide. RT-PCR was done use the fish treated with polyinosinic acid-polycytidylic acid (poly I:C) and grass carp haemorrhagic virus to determine the tissue distrubution of the gcIFN. The results reveal that IFN is highly expressed in brain, heart,and head kindey although its expression can be also found in other tissues. The gcIFN was subcloned into yeast expression vector pYES2 (to generate a recombinant plasmid IFN-pYES2) and then transformed into S.cerevisiae strain INVScl. The recombinant yeast cells were selected on a uracil-deficient medium and the GC-IFN protein was deduced by galcose. The purified gcIFN protein could induce the expression of Mx protein in the grass carp ZC7901 cell line.In the part two, we have cloned and characterized the full-length cDNA of NF45 in grass carp. The grass carp NF45 cDNA of 1563bp contains a short 5'UTR of 24bp, a 3'UTR of 375bp and an open reading frame of 1164 bp coding for a protein of 387 amino acids with a predicted molecular mass of 42.8 kDa. Meanwhile, RT-PCR was optimized to estimate the expression level of NF45 in grass carp. The results showed that NF45 was constitutively expressed in most selected tissues, including head kidney, spleen, heart, brain, liver, and gill, although low levels were observed in spleen, liver and gill. The ubiquitous expression of NF45 is consistent with a postulated role in gene regulation at the level of transcription. Stimulating the fish with PHA significantly up-regulated the expression of NF45 in most tissues examined, which potentially indicated that NF45 was involved in the immune responses triggered by PHA. Our work will help to elucidate the multifaceted contributions of NF45 to the gene regulations in fish.Interferon is a cytokine that play an important role when body defense against viruses. Interferon system have a major role in the first line of the defence. The antiviral effect of fish type I IFN is exerted through binding to the IFN-a/b-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and otherantiviral proteins. NF45 gene which also called interleukin binding enhancer factor, regulate the IL-2 gene transcription via interaction with the antigen receptor response element which located in the IL-2 promoter area. NF-45 is an important transcription factor regulating the IL-2 expression. Fish connects the high vertebrate and low invertebrate, and plays a specific role in evolution. The current research in this paper help to further known about these two gene and known how to defence against the fish dieases.