MOLECULAR CLONING AND CHARACTERIZATION OF <I>Nub</I>1 GENE FROM CRUCIAN CARP <I>CARASSISUS AURATUS</I>

Abstract

Interferon(IFN) system is the first line of defense against invading virus pathogens in vertebrate.IFN-α/β plays its critical role through a subset of IFN-stimulated genes(ISGs) to directly inhibit viral replication,cell growth,control apoptosis and mediate a variety of immunomodulatory effects.More than 300 ISGs have been identified by oligonucleotide array,and some of them such as Mx,dsRNA dependent protein kinase R(PKR),2',5'-oligoadenylate synthetase(2—5 OAS)/RNase L and RNA-specific adenosine deaminase(ADAR) have been known to affect virus replication,transcription or cell growth.NEDD8 Ultimate Buster-1(NUB1) is identified as an IFN inducible gene recently,and over-expression of human NUB1 results in inhibition of cell growth.Despite the significant achievements in mammals,relative little has been known concerning fish IFN system genes since the discovery of fish IFN-like activity in 1965.More recently,much effort has been made in isolating virally induced genes in fish,including genes involved in fish IFN system,and a subtractive cDNA library was constructed with mRNAs obtained from crucian carp(Carassius auratus L.) blastulae embryonic cells(CAB) infected by UV-inactivated grass carp hemorrhage virus(GCHV) and control cells.In this study,an EST homologous to mammalian gene Nub1 was obtained from this cDNA library.Furthermore,the full-length cDNA of Carassius auratus Nub1(CaNub1) was obtained by RACE-PCR and its expression was subsequently characterized by semi-quantitative RT-PCR.The full-length cDNA of CaNUB1 is 2298 bp,which encodes a protein of 589 amino acids.Structurally,the putative CaNUB1 protein contains a ubiquitin like(UBL) motif in N terminus and two ubiquitin associated domains(UBA) in C terminus,showing 41%—45% homology to known counterparts from mammals,birds and amphibian.Expression analysis showed that Poly I:C and LPS were able to upregulate the expression of CaNUB1,indicating that CaNUB1 play some roles in fish antiviral immune response.