Cloning And Expression Of The Interferon-α Gene From Grass Carp And Its Effects On Fish Pathogenic Rhabdoviruses

After sequencing the zebrafish genome and along with the further researching on the expressed sequence tag (EST), the interferon of zebrfish was cloned in 2003 .To further evaluate the clinical impact of fish recombinant interferon, the grasscarp (C. idellus) IFN-α (GcIFN-α) gene was cloned from the cDNA library by PCR, and expressed in Escherichia coli expression system and assayed the anti-viral activity in the study.A pair of degenerate primers were designed based on conserved regions in fish interferon gene in DDBJ/GenBank. After amplification from cDNA library, the PCR product was inserted into pGEM T-easy vector and sequenced. Then, a pair of primers to express GcIFN-a was designed. The PCR product cloned by a standard PCR was subcloned into E.coli expression vector pQE30. The cells were harvested and total E.coli proteins were detected by SDS-PAGE to select expression strains. The rGcIFN-a was centrifuged, sonicated, then its renaturated and its effects on vesicular stomatitis virus, spring viremia of carp virus, and infectious hematopoietic necrosis virus were surveyed in homologous and heterologous cells by inhibition of the cytopathic effect.The cloned GcIFN-a gene encoded 158 amino acids, and had a potential N-glycosylation site and one pair of cysteines to form an intrachain disulfide bridge within site 1 and site 97. Comparison of GcIFN-a amino acids with other fish Type I IFN showed the identical ratio to be 30.6%-72.2%. The recombinant protein with 6 consecutive histidine residues was expressed with molecular weight of 20.2kDa. The proportion of rGcIFN-a in total E.coli proteins is approximately 42%. The anti-virus assay suggested that rGcIFN-a could inhibit these pathogenic rhabdoviruses on C. idellus ovaries and Epithelioma papulosum cyprini cell lines, but had no anti-VSV activity in chicken embryo fibroblast.