Molecular Cloning And Functional Study Of STAT1 From Black Carp

Signal transducer and transcriptional activator/STAT 1 plays an significant role in Janus kinase(JAK)-STAT signaling of human and mammalian.After STAT1 is phosphorylated by activated JAK1,it will dimerize and enter the nucleus exercising function.Black carp grows very fast and occupies a large proportion of the economic fish farmed in China,and its production speed is relatively fast,but it is susceptible to external pathogens during the juvenile period,which is always a problem for the black carp cultivation.Innate immunity is the first line of defense for host cells to resist the invasion of pathogenic microorganisms,so the study of disease resistance mechanism in black carp innate immune will promote the solution of this problem.In this paper,the function of black carp immune factor STAT1 was investigated.two STAT1 homologous genes(bcSTAT1a and bcSTAT1b)of black carp were cloned and identified.The CDS of bcSTAT1 a includes 2400 nucleotides and the predicted protein consists of 800 amino acids,bcSTAT1 b includes 2157 nucleotides and consists of 719 amino acids.The isoelectric points of bcSTAT1 a and bcSTAT1 b are 5.65 and 5.58 respectively.In the qPCR experiment,we found that under the stimulation of ploy(I:C),lipopolysaccharide(LPS),grass carp reovirus(GCRV)and interferon(Interforen/IFN),the transcription levels of bcSTAT1 a and bcSTAT1 b in host cells(MPK)were significantly increased,but by comparison,we found that the transcription level of bcSTAT1 b was significantly higher than that of bcSTAT1 a under the stimulation of different foreign species.In the immunofluorescence and immunoblot assays,we found that bcSTAT1 a and bcSTAT1 b are expressed in the cytoplasm and nucleus,and bcSTAT1 a and bcSTAT1 b migrated about 95 KDa and 82 KDa in HEK293 T and EPC cells.The interaction between bcSTAT1 a andbcSTAT1a,bcSTAT1 b and bcSTAT1 b,and bcSTAT1 a and bcSTAT1 b was detected by immunoprecipitation(co-ip);and the non-denaturing polyacrylamide gel electrophoresis(SDS-PAGE)data show that bcSTAT1 a and bcSTAT1 b may form homodimer/heterodimer in vivo,which is similar to mammals.In a dual fluorescence reporter system,EPC cells transfected with bcSTAT1 a and bcSTAT1 b showed dose-dependent inducibility to zebrafish interferon promoter 3(DrIFN?3)and black carp interferon promoter(bcIFN?).The results of plaque assay showed that overexpression of bcSTAT1 a and bcSTAT1 b in EPC cells enhanced antiviral activity against GCRV compared with the control group.Our data support the conclusion that both bcSTAT1 a and bcSTAT1 b play important roles in host antiviral innate immune activation initiated by GCRV.