Cloning And Expression Analysis Of Immune Genes In Crucian Carp(Carassius Auratus Gibelio) And The Immune Protection Of Recombinant Ifnc Protein Against Cyprinid Herpesvirus 2

Crucian carp(Carassius auratus gibelio)is a popular food fish in Asia,and CyHV-2(Cyprinid Herpesvirus 2)is the only known viral pathogen for crucian carp.IκBα(NF-κB inhibitor),Rab21(Rab GTPase21)play important roles in innate immunity.Previously,they were identified by suppression subtractive hybridization as differentially expressed immune-related genes in crucian carp infected with Cyprinid Herpesvirus 2(CyHV-2).Here,full-length IκBα,Rab21 cDNAs cloned from crucian carp showed high homologies with transcripts in other bony fish.IκBα spanned over 933 bp and encoded a polypeptide containing 310 amino acids(KJ784543).Rab21 spanned over 648 bp and encoded a polypeptide containing 215 amino acids(KJ784545).Normally expressed at low levels in all crucian carp tissues examined,these genes were significantly upregulated in kidneys from 6–24 h post-infection with CyHV-2(P < 0.01).These data suggested IκBa,Rab21 might be antiviral with potential involvement,IκBα and Rab21 could serve as immune markers for host immune response to CyHV-2 chronic infection.Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms.In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge,a partial interferon transcript was identified to be significantly upregulated in the kidney of infected fish,which was named as crucian carp IFNc(ccIFNc).The complete ORF of ccIFNc was further determined by RACE technique,which spanned over 546 bp and encoded a polypeptide containing 182 amino acids(BankIt2033924).Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms.Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen,gill,kidney,and muscle.Following challenge with CyHV-2,the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues,especially in the spleen and gill with increased folds of 436 and 158,respectively.The intramuscular(i.m.)injection of a eukaryotic expression plasmid encoding ccIFNc(pEGFP-cIFNc)resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly.In summary,our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.