| Grass carp(Ctenopharyngodon idellus)is one of the most important freshwater cultured fishes in China.Its meat is tender,delicious,fast growing and easy to breed.It is favored by the majority of consumers and farmers.It is an important part of China’s bulk freshwater economic fish culture.However,Ctenopharyngodon hemorrhagic disease caused by grass carp reovirus(GCRV)seriously restricts the healthy development of grass carp breeding.Viperin is a broad-spectrum antiviral protein,which plays an important role in fish innate immunity.However,the mechanism of Viperin’s antiviral function is different for different viruses.The antiviral mechanism of Ctenopharyngodon idellus Viperin has not been reported.Therefore,the rapid amplification of c DNA ends(RACE)method was used to clone the gene of Ctenopharyngodon idellus(CiViperin).The structure of CiViperin gene was analyzed by bioinformatics,and the anti GCRV function of Viperin was preliminarily studied.So as to lay a foundation for further exploring the function of Viperin in Ctenopharyngodon idellus.In this study,the c DNA sequence of Viperin gene of Ctenopharyngodon idellus was cloned by race technology,and the structural characteristics,evolutionary relation ship and gene collinearity of CiViperin gene were analyzed by bioinformatics methods.The expression of Viperin in normal tissues and the related immune tissues of Cte nopharyngodon idelluschallenged with GCRV,Poly(I:C)and LPS were analyzed by fluorescence quantitative PCR.Objective to construct the expression vector of pEGFPN1-Flag-Viperin and investigate the preliminary function of Viperin against GCRV in CIK cells.The results showed that the full length c DNA of Viperin gene was 1885 bp,including29 bp 5′ UTR,776 bp 3′ UTR,1080 bp ORF,encoding 359 amino acids;the molecular weight of CiViperin protein predicted was 41.56 k D by Ex PASY,and the theoretical isoelectric point was 8.56;the genome structure of Viperin was composed of 6 exons and 5 introns.SMART domain prediction showed that CiViperin contained a classical domain of El P3,and CiViperin 3D was composed of 12 α-helices and 14 β-folds.Phylogenetic tree showed that the amino acid sequence of CiViperin was clustered with that of fish Viperin,and had high similarity and homology with that of black carp.CiViperin gene was expressed in liver,spleen,kidney,head kidney,gill,skin,muscle,intestine,brain and heart of healthy Ctenopharyngodon idellus,and its expression level was higher in gill and spleen,followed by brain and heart;after GCRV challenge,CiViperin gene expression level in liver,spleen,kidney and head kidney showed a trend of first increase and then decrease,and the highest expression level was in liver At 120 h after GCRV challenge(P<0.05),the highest expression in spleen appeared at 168 h after GCRV challenge(P<0.05).The highest expression was found in the kidney 24 h after GCRV challenge CiViperin gene expression level in head kidney tissue was the highest at 72 h after challenge(P<0.05);CiViperin gene expression level in liver,spleen,kidney and head kidney tissue of grass carp first showed an upward trend after poly(I:C)and LPS stimulation,and then decreased with time.In the middle period,poly(I:C)and LPS stimulation could cause liver injury CiViperin gene expression level was up-regulated in GCRV,spleen,kidney and head kidney tissues,and there was significant difference compared with the control group(P<0.05).CiViperin gene expression level decreased slightly with the extension of detection time point.The m RNA expression of VP7 in CIK cells detected by CiViperin overexpression showed that the m RNA expression of VP7 in GCRV decreased significantly from 12 h to 24 h The expression level of VP7 gene in GCRV was significantly higher than that in control group(P<0.05),which indicated that Viperin could inhibit the transcription and expression of VP7 gene in GCRV.In conclusion,the structure of Viperin gene of Ctenopharyngodon idellushas a classic El P3 domain,which is similar to that of fish Viperin in evolution and has high homology;Viperin gene of Ctenopharyngodon idellusis expressed in different tissues,and its expression is up-regulated under the stimulation of GCRV,Poly(I:C)and LPS,which has inhibitory effect on VP7,which is responsible for the adsorption of GCRV to host cells.The results of this study laid a theoretical foundation for further study on the mechanism of Viperin in the anti GCRV effect of Ctenopharyngodon idellusis. |
